Novel indocyanine compound, synthesis method and purification method thereof, diagnostic composition using the indocyanine compound, and device for measuring biokinetics and device for visualizing circulation using the diagnostic composition

ABSTRACT

The present invention aims at providing a novel indocyanine compound solving problems of conventionally used indocyanine green, such as solubility in water or physiological saline, a synthesis method and a purification method thereof, and a diagnostic composition including the novel indocyanine compound. Further, provided are a method for evaluating biokinetics of the novel indocyanine compound and a device for measuring biokinetics, and a method and a device for visualizing circulation of fluid such as blood in a living body, which utilize the diagnostic composition. Also, found are a novel indocyanine compound in which a hydrophobic moiety in a near-infrared fluorescent indocyanine molecule is included in a cavity of a cyclic sugar chain cyclodextrin to cover the hydrophobic moiety in the indocyanine molecule with the glucose, and a synthesis method and a purification method thereof. Furthermore, found are a method for fluorescence-imaging an organ other than liver by intravenous administration, a method for evaluating biokinetics of the novel indocyanine compound, a device for measuring biokinetics, and a method and a device for visualizing circulation of fluid such as blood in a living body, utilizing the diagnostic composition including the novel indocyanine compound.

TECHNICAL FIELD

The present invention relates to a novel indocyanine compound which is agreen pigment useful for medical diagnostic technologies, medicalsurgical technologies, scientific measurement and analysis technologies,printing technologies, writing technologies, coating technologies,dyestuffs technologies and dyeing technologies, and has a propertycapable of emitting near-infrared fluorescence, a synthesis method and apurification method thereof, and a diagnostic composition. Moreparticularly, the present invention relates to a cyclic sugar chaincyclodextrin-bonded indocyanine compound which is a green pigment andhas a property capable of emitting near-infrared fluorescence, asynthesis method and a purification method thereof, and a diagnosticcomposition using the indocyanine compound, a device for measuringbiokinetics and a device for visualizing a circulation using thediagnostic composition.

BACKGROUND ART

Indocyanine compounds which are green pigments and emit near-infraredfluorescence have hitherto been synthesized, and they have variousapplications such as pigments for dyeing used in surgeries of vitreousbody of the eye, pigments used in medicines for testing liver functions,pigment used in medicines for testing circulatory functions, pigmentsused for surgical operations, and near-infrared fluorescent compoundsused for surgical operation in medical fields; dyeing of proteins orsugars and compounds for fluoresceination in scientific fields; andpigments in printing technologies. Of these indocyanine compounds, acompound which is called as an indocyanine green (hereinafter referredto as “ICG”) has been used as a medicine for testing liver functions orcirculatory functions for nearly 50 years. ICG has recently been usedfor medical operations or medical diagnoses, utilizing its property ofhigh light-permeability from a biological tissue, by topicallyadministrating ICG to a body such as a blood vessel, lymph vessel,brain, eye, stomach, breast, esophagus, skin or another site andobserving the near-infrared fluorescence of ICG, which is a trial runyet though, as the application of ICG other than the medicines fortesting liver functions or circulatory function (Non Patent Document 1).

PRIOR ART TECHNICAL DOCUMENTS Non Patent Document

-   Non Patent Document 1: “All of ICG fluorescence Navigation Surgery”,    supervised and edited by Mitsuo Kusano, Intermedica Co., Ltd,    (published on November, 2008).-   Non Patent Document 2: URL    (https://www.daiichisankyo.co.jp/med/contents/di/dg2/pi/pdf/    pi_dg2_0909.pdf) of Internet Homepage in which an attached document    to “(trademark) Diagnogreen for injection 25 mg” (Daiichi Sankyo    Company, Limited) is provided.-   Non Patent Document 3: R. C. Benson, H. A. Kues, Phys. Med. Biol.,    23, 159-163 (1978).-   Non Patent Document 4: S. Yoneyama, T. Saito, Y. Komatsu, I.    Koyama, K. Takahashi, J. Duvoll-Young, IOVS, 37, 1286-1290 (1998).-   Non Patent Document 5: Y. Ye, W. P. Li, C. J. Anderson, J.    Kao, G. V. Nikiforovich, S. Achilefu, J. Am. Chem. Soc., 125,    7766-7767 (2003).-   Non Patent Document 6: K. Teranishi and S. Tanabe, ITE Letters on    Batteries, New Technologies & Medicine, 1, 53-60 (2000).-   Non Patent Document 7: MICHAEL O'SHAUGHNESSY et al: 1994 Wiley-Liss,    Inc MICROSURGERY 15: 40 S 412 1994.-   Non Patent Document 8: RALPH J. P. M et al: 1997 Wiley-Liss, Inc.    MICROSURGERY 17: 402-408 1996.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

A green chromophore (a chemical structure necessary for exhibitinggreen) and a near-infrared fluorophore (a chemical structure necessaryfor exhibiting near-infrared fluorescence) of ICG, however, arehydrophobic, and thus sulfonyl groups are bonded to the side-chainterminus to render them water-soluble. This gives many problemsdescribed below to the conventional ICG.

When ICG preparation is used in medical applications, distilled water isgenerally added to 25 mg of ICG in an amount of about 5 mL to 10 mL, andthe ICG is dissolved by vibration agitation. If the ICG is notcompletely dissolved, vomiturition, nausea, fever or a shock-likesymptom may sometimes occur (Non Patent Document 2). Additionally, it isimpossible to initially dissolve it in another aqueous solution such asphysiological saline because of insolubilization (Non Patent Document2).

ICG is water-soluble because sulfonyl groups are bonded thereto, asdescribed above, but it has a surface activity because of its chemicalstructure in which there are many hydrophobic hydrocarbon groups, andthus it has a property of adsorbing to lipids. When it is administeredto a biological tissue such as a blood vessel or an organ, accordingly,it adheres to the injected site, it is leaked by mistake or it is flownbackward, whereby it may sometimes adhere to an undesired biologicaltissue. ICG adhering to the biological tissue cannot be easily removedfrom the biological tissue by wiping it away or sucking it, leading to apossibility in which a surgical operation or a medical diagnosis isinterfered.

ICG has a property of assembling molecules in an aqueous solution. ICG,thus, has a low fluorescence intensity in an aqueous solution, thoughthe property is one of primary factors (Non Patent Documents 3 and 4).

In addition, ICG becomes insolubilized as time advances after it isdissolved in water, and thus it is difficult to store it in the state ofan aqueous solution for a long term. In addition, at a low temperature,freeze preservation thereof promotes the insolubilization.

ICG preparation includes 5% or less NaI, and has a defect in which itmay cause iodine hypersensitivity (Non Patent Document 2).

When ICG is intravenously injected, it is promptly accumulated in aliver and excreted through the liver, and accordingly, fluorescenceimaging of another organ such as a kidney, ureter, bladder, urethra,heart or lung is difficult.

In addition, when ICG is intravenously injected, it transfers with bloodand it transfers a little to peripheral tissues, and accordingly it isdifficult to observe the transfer to an interstitial tissue.

The present invention aims at solving the problems of the conventionalICG described above, in other words, the present invention aims atproviding a novel indocyanine compound which is a green pigment andexhibits near-infrared fluorescence, characterized by a high solubilityin water or physiological saline, easy removal from a biological tissue,a low molecule association in an aqueous solution, a high near-infraredfluorescence intensity in an aqueous solution, and fluorescence imagingof an organ other than liver such as kidney, ureter, bladder, urethra,heart or lung. The present invention further provides a chemicalsynthesis method and a purification method of the novel indocyaninecompound having the features described above. To provide a diagnosticcomposition including the novel indocyanine compound is also a problemto be solved by the invention. Furthermore, the present invention aimsat providing a device for measuring biokinetics capable of evaluatingbiokinetics of the novel indocyanine compound, concerning a horizontalequilibrium in a living body, and a method and device for visualizing acirculation of blood, lymph fluid, urine or other fluid in a livingbody, utilizing the diagnostic composition.

Means of Solving the Problem

The present inventors have repeated painstaking studies in order tosolve the problems described above; as a result, they have found acompound exhibiting near-infrared fluorescence, which can be used in asurgical operation or medical diagnosis utilizing the property ofexhibiting near-infrared fluorescence, and have solved the problems ofICG described above.

The present inventors have found a novel indocyanine compound which is agreen pigment characterized by a high solubility in water orphysiological saline, easy removal from a biological tissue, a lowmolecule association in an aqueous solution, a high near-infraredfluorescence intensity in an aqueous solution, and exhibition ofnear-infrared fluorescence; and have completed the present invention.Further, they have found a chemical synthesis method and a purificationmethod of the novel indocyanine compound, and have completed the presentinvention. Furthermore, they have provided a diagnostic compositionincluding the novel indocyanine compound. Still further, they haveprovided a method for fluorescence imaging an organ other than livereven in intravenous administration by utilizing the diagnosticcomposition. Still further, they have provided a method for evaluatingbiokinetics of the novel indocyanine compound, concerning a horizontalequilibrium in a living body and a device for measuring biokinetics, anda method and a device for visualizing circulation of blood, lymph fluid,urine or other fluid in a living body.

A first novel indocyanine compound of the present invention is acompound in which a cyclic sugar chain cyclodextrin is covalently bondedto a green chromophore (a chemical structure necessary for exhibitinggreen) and a near-infrared fluorophore (a chemical structure necessaryfor exhibiting near-infrared fluorescence) of ICG. Further, a secondnovel indocyanine compound of the invention is a novel indocyaninecompound which is characterized in that a naphthyl moiety which is ahydrophobic moiety of an indocyanine structure is included in a cavityof a cyclodextrin to cover a naphthyl moiety which is a hydrophobicmoiety with a hydrophilic glucose group, thereby three-dimensionallyhydrophilizing many regions of the indocyanine molecule structure. ICGis characterized by having a surfactant-like property in which it hasboth hydrophobic moieties and hydrophilic property caused by sulfonylgroups; whereas, the compound of the invention is characterized byhaving no surfactant-like property because the hydrophobic moieties inits molecule is covered with the cyclodextrins. In more detail, thepresent inventions are:

<1> A cyclodextrin-bonded indocyanine compound in which an indocyanineis covalently bonded to a cyclic sugar chain cyclodextrin, which isrepresented by the following chemical formula 1:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are a hydrogen atom, analkyl group, an aryl group, a halogen atom, an alkoxyl group, an aminogroup, a carboxyl group, a formyl group, a sulfonyl group, a sulfonicacid group, a phosphate group, an alkyloxycarbonyl group, anaryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, ora heterocyclic ring; when a hydrogen ion on the substituents (thecarboxylic acid, the sulfonic acid and the phosphoric acid) dissociates,a metal ion such as a sodium ion, a potassium ion, a magnesium ion or acalcium ion may be substituted for the hydrogen ion; the amino group isalso selected from primary, secondary, tertiary and quaternary groups (asubstituent bonded to the nitrogen atom includes an alkyl group, and thelike); a cyclic structure of CH₂, CH₂CH₂, CH₂CH₂CH₂ or CH₂CH₂CH₂CH₂ isselected as the groups R₈ and R₉; and a functional group of an alkylgroup, an aryl group, a halogen atom, an alkoxy group, an amino group, acarboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group,a phosphate group, an alkyloxycarbonyl group, an aryloxycarbonyl group,an alkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring isalso substituted for the hydrogen atom on the alkyl groups.

<2> A cyclodextrin-bonded indocyanine compound in which at least a partof a naphthyl group of an indocyanine is included in a cavity of acyclodextrin, represented by the following chemical formula 2:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are a hydrogen atom, analkyl group, an aryl group, a halogen atom, an alkoxyl group, an aminogroup, a carboxyl group, a formyl group, a sulfonyl group, a sulfonicacid group, a phosphate group, an alkyloxycarbonyl group, anaryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, ora heterocyclic ring; when a hydrogen ion on the substituents (thecarboxylic acid, the sulfonic acid and the phosphoric acid) dissociates,a metal ion such as a sodium ion, a potassium ion, a magnesium ion or acalcium ion may be substituted for the hydrogen ion; the amino group isalso selected from primary, secondary, tertiary and quaternary groups; acyclic structure of CH₂, CH₂CH₂, CH₂CH₂CH₂ or CH₂CH₂CH₂CH₂ is alsoselected as the groups R₈ and R₉; and a functional group of an alkylgroup, an aryl group, a halogen atom, an alkoxy group, an amino group, acarboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group,a phosphate group, an alkyloxycarbonyl group, an aryloxycarbonyl group,an alkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring isalso substituted for the hydrogen atom on the alkyl groups.

<3> The cyclodextrin-bonded indocyanine compound in which an indocyanineis covalently bonded through an amide bond to a cyclic sugar chaincyclodextrin, represented by the chemical formula 1, wherein thecompound is a cyclodextrin-bonded indocyanine compound represented bythe following chemical formula 3:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are a hydrogen atom, analkyl group, an aryl group, a halogen atom, an alkoxyl group, an aminogroup, a carboxyl group, a formyl group, a sulfonyl group, a sulfonicacid group, a phosphate group, an alkyloxycarbonyl group, anaryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, ora heterocyclic ring; when a hydrogen ion on the substituents (thecarboxylic acid, the sulfonic acid and the phosphoric acid) dissociates,a metal ion such as a sodium ion, a potassium ion, a magnesium ion or acalcium ion may be substituted for the hydrogen ion; the amino group isalso selected from primary, secondary, tertiary and quaternary groups; mand n are an integer of 1 or more and 6 or less; a cyclic structure ofCH₂, CH₂CH₂, CH₂CH₂CH₂ or CH₂CH₂CH₂CH₂ is also selected as the groups R₈and R₉; and a functional group of an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, a phosphategroup, an alkyloxycarbonyl group, an aryloxycarbonyl group, analkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring isalso substituted for the hydrogen atom on the alkyl groups.

<4> The cyclodextrin-bonded indocyanine compound in which an indocyanineis covalently bonded through an amide bond to a cyclic sugar chaincyclodextrin, represented by the chemical formula 2, wherein thecompound is a cyclodextrin-bonded indocyanine compound represented bythe following chemical formula 4:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are a hydrogen atom, analkyl group, an aryl group, a halogen atom, an alkoxyl group, an aminogroup, a carboxyl group, a formyl group, a sulfonyl group, a sulfonicacid group, a phosphate group, an alkyloxycarbonyl group, anaryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, ora heterocyclic ring; when a hydrogen ion on the substituents (thecarboxylic acid, the sulfonic acid and the phosphoric acid) dissociates,a metal ion such as a sodium ion, a potassium ion, a magnesium ion or acalcium ion may be substituted for the hydrogen ion; the amino group isalso selected from primary, secondary, tertiary and quaternary groups; mand n are an integer of 1 or more and 6 or less; a cyclic structure ofCH₂, CH₂CH₂, CH₂CH₂CH₂ or CH₂CH₂CH₂CH₂ is also selected as the groups R₈and R₉; and a functional group of an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, a phosphategroup, an alkyloxycarbonyl group, an aryloxycarbonyl group, analkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring isalso substituted for the hydrogen atom on the alkyl groups.

<5> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 3, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 5:

wherein m, n, p and q are an integer of 2 or more and 6 or less; r is aninteger of 5 or more and 7 or less; s is an integer of 0 or more and 4or less; and R is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, analkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonylgroup, an arylcarbonyl group or a heterocyclic ring.

<6> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 4, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 6:

wherein m, n, p and q are an integer of 2 or more and 6 or less; r is aninteger of 5 or more and 7 or less; s is an integer of 0 or more and 4or less; and R is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, analkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonylgroup, an arylcarbonyl group or a heterocyclic ring.

<7> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 3, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 7:

wherein m and n are an integer of 2 or more and 6 or less; r is aninteger of 5 or more and 7 or less; s is an integer of 0 or more and 4or less; and R is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, analkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonylgroup, an arylcarbonyl group or a heterocyclic ring.

<8> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 4 which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 8:

wherein m and n are an integer of 2 or more and 6 or less; r is aninteger of 5 or more and 7 or less; s is an integer of 0 or more and 4or less; and R is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, analkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonylgroup, an arylcarbonyl group or a heterocyclic ring.

<9> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 3, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 9:

wherein m and n are an integer of 2 or more and 6 or less; r is aninteger of 5 or more and 7 or less; s is an integer of 0 or more and 4or less; and R is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, analkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonylgroup, an arylcarbonyl group or a heterocyclic ring.

<10> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 4, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 10:

wherein m and n are an integer of 2 or more and 6 or less; r is aninteger of 5 or more and 7 or less; s is an integer of 0 or more and 4or less; and R is a hydrogen atom, an alkyl group, an aryl group, ahalogen atom, an alkoxy group, an amino group, a carboxyl group, aformyl group, a sulfonyl group, a sulfonic acid group, analkyloxycarbonyl group, an aryloxycarbonyl group, an alkylcarbonylgroup, an arylcarbonyl group or a heterocyclic ring.

<11> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 3, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 11:

wherein s is an integer of 0 or more and 4 or less; and R is a hydrogenatom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, anamino group, a carboxyl group, a formyl group, a sulfonyl group, asulfonic acid group, an alkyloxycarbonyl group, an aryloxycarbonylgroup, an alkylcarbonyl group, an arylcarbonyl group or a heterocyclicring.

<12> The cyclodextrin-bonded indocyanine compound represented by thechemical formula 4, which is a cyclodextrin-bonded indocyanine compoundrepresented by the following chemical formula 12:

wherein s is an integer of 0 or more and 4 or less; and R is a hydrogenatom, an alkyl group, an aryl group, a halogen atom, an alkoxy group, anamino group, a carboxyl group, a formyl group, a sulfonyl group, asulfonic acid group, an alkyloxycarbonyl group, an aryloxycarbonylgroup, an alkylcarbonyl group, an arylcarbonyl group or a heterocyclicring.

<13> A chemical synthesis method of the cyclodextrin-bonded indocyaninecompound represented by any one of the chemical formulae described above(chemical formulae 1, 3, 5, 7, 9 and 11), including: (1) a step ofmixing an indocyaninecarboxylic acid compound with an aminocyclodextrinin a medium, and (2) a step of a dehydration-condensation reaction ofthe mixture by addition of a dehydration-condensing agent thereto.

<14> A chemical synthesis method of the cyclodextrin-bonded indocyaninecompound represented by any one of the chemical formulae described above(chemical formulae 2, 4, 6, 8, 10 and 12), including a step ofsubjecting the cyclodextrin-bonded indocyanine compound represented byany one of the chemical formulae described above (chemical formulae 1,3, 5, 7, 9 and 11) to an inclusion reaction in water.

<15> A purification method of the cyclodextrin-bonded indocyaninecompound represented by any one of the chemical formulae described above(chemical formulae 1 to 12), including a step of purifying the compoundby a column chromatography eluting it with a medium including HCl.

<16> A diagnostic composition which is an aqueous solution including thecyclodextrin-bonded indocyanine compound represented by any one of thechemical formulae 1 to 12, and is used by infusing it into a body.

<17> The diagnostic composition according to the item <16>, which doesnot substantially include iodine.

<18> A device for measuring biokinetics of a cyclodextrin-bondedindocyanine compound including:

-   excitation light-irradiating means of irradiating excitation light    to the cyclodextrin-bonded indocyanine compound in a part of a    living body to which the diagnostic composition according to item    <16> or <17> is administered;-   fluorescence intensity-measuring means of measuring an intensity of    fluorescence emitted by the cyclodextrin-bonded indocyanine compound    which has been excited by the excitation light-irradiating means;    and-   biokinetics-calculating means of calculating a transfer speed of the    cyclodextrin-bonded indocyanine compound in the part of the living    body into and/or out of interstitial tissue fluid by obtaining a    change with time in the fluorescence intensity from the fluorescence    intensities acquired by the fluorescence intensity-measuring means    with time.

<19> A device for visualizing circulation including:

-   excitation light-irradiating means of irradiating excitation light    to a cyclodextrin-bonded indocyanine compound in a part of a living    body to which the diagnostic composition according to item <16> or    <17> is administered;-   fluorescence-imaging means of obtaining distribution state data of    the cyclodextrin-bonded indocyanine compound in the living body by    two-dimensionally acquiring a fluorescence intensity emitted by the    cyclodextrin-bonded indocyanine compound which has been excited by    the excitation light-irradiating means;-   morphological imaging means of obtaining morphological data of the    part of the living body by two-dimensionally acquiring an intensity    of light having a wavelength other than the fluorescence wavelength    emitted by the cyclodextrin-bonded indocyanine compound; and-   displaying means of displaying a distribution state of the    cyclodextrin-bonded indocyanine compound in the part of the living    body by overlapping the morphological data obtained by the    morphological imaging means with the distribution state data    obtained by the fluorescence-imaging means.

<20> The device for visualizing circulation according to item <19>9,wherein the displaying means displays an area in which a distributionquantity of the cyclodextrin-bonded indocyanine compound is lower than apredetermined standard in the part of the living body as a necrosisarea.

<21> The device for measuring biokinetics according to item <18>, whichfurther includes tumescence progression-predicting means of predicting adegree of tumescence which will advance in future from the transferspeed into or out of the interstitial tissue fluid in the part of theliving body.

<22> The device for measuring biokinetics according to item <21>,wherein the tumescence progression-predicting means predicts a degree oftumescence progression corresponding to the transfer speed into theinterstitial tissue fluid after a predetermined time course until thecyclodextrin-bonded indocyanine compound is dispersed in blood in awhole body from the administration of the diagnostic composition.

<23> A device for measuring biokinetics including:

-   excitation light-irradiating means of irradiating excitation light    to a cyclodextrin-bonded indocyanine compound in a part of a living    body or a control moiety to which the diagnostic composition    according to item <16> or <17> is administered;-   fluorescence intensity-measuring means of measuring an intensity of    fluorescence emitted by the cyclodextrin-bonded indocyanine compound    which has been excited by the excitation light-irradiating means;-   biokinetics-calculating means of calculating a transfer speed of the    cyclodextrin-bonded indocyanine compound in the part of the living    body into and/or out of interstitial tissue fluid by obtaining a    change with time in the fluorescence intensity from the fluorescence    intensities acquired by the fluorescence intensity-measuring means    with time; and-   tumescence progression-predicting means of predicting a degree of    tumescence which will advance in future from the transfer speed into    or out of the interstitial tissue fluid in the part of the living    body,-   wherein the tumescence progression-predicting means is means of-   obtaining a relationship between a volume of blood flowing in the    control moiety and a volume of blood flowing in the part of the    living body, from a change in the fluorescence intensity in the part    of the living body and a change in the fluorescence intensity in the    control moiety up to a predetermined time until the    cyclodextrin-bonded indocyanine compound is dispersed in blood in a    whole body after the administration of the diagnostic composition,-   calculating a degree of the change in the fluorescence intensity in    the part of the living body comparing with the change in the    fluorescence intensity in the control moiety, using the relationship    after the predetermined time course, and-   predicting a degree of tumescence progression corresponding to the    degree of the change calculated above.

<24> A device for measuring biokinetics including:

-   excitation light-irradiating means of irradiating excitation light    to a cyclodextrin-bonded indocyanine compound in an organ of a    living body to which the diagnostic composition according to item    <16> or <17> is administered;-   fluorescence intensity-measuring means of measuring an intensity of    fluorescence emitted by the cyclodextrin-bonded indocyanine compound    which has been excited by the excitation light-irradiating means;    and-   biokinetics-calculating means of evaluating biokinetics of the    cyclodextrin-bonded indocyanine compound in the organ from the    fluorescence intensity acquired by the fluorescence    intensity-measuring means with time.

<25> The device for measuring biokinetics according to item <24>,wherein the organ is any one of kidney, ureter, bladder and urethra.

Effect of the Invention

A compound can be provided from the cyclodextrin-bonded indocyaninecompound represented by the chemical formula 1 or chemical formula 2 ofthe present invention which is a green pigment and exhibitsnear-infrared fluorescence characterized in that a solubility in wateror physiological saline is higher than that of ICG, it can be easilyremoved from a biological tissue, a molecule association is low in anaqueous solution, a near-infrared fluorescence intensity is high in anaqueous solution, fluorescence imaging of organs other than liver can beperformed, and it includes no iodine. In addition, the synthesis methodof the cyclodextrin-bonded indocyanine compound of the present inventioncan provide a useful synthesis of the cyclodextrin-bonded indocyaninecompound. Additionally, the purification method of thecyclodextrin-bonded indocyanine compound of the present invention canprovide a useful purification of the cyclodextrin-bonded indocyaninecompound. Further, the cyclodextrin-bonded indocyanine compound of thepresent invention shows a sufficient solubility even if it does notinclude iodine, and, accordingly, it can provide a diagnosticcomposition including no iodine which leads to iodine hypersensitivity.This diagnostic composition shows a biohehavior different from adiagnostic composition including conventional ICG alone, and thus canprovide various useful devices utilizing the properties.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of adsorption tests of ICG and the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to human skin. It shows a state immediately after application of 1 mMaqueous solution (0.03 mL) including ICG or the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to an arm (a left photograph), a state immediately after washing it withwater 5 minutes later (a middle photograph), and a state immediatelyafter rubbing it and washing it with water (a right photograph), whereina point at a right side is a marker by a red pen.

FIG. 2 shows results of adsorption tests of ICG and the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to a cellulose fiber. It shows a state immediately after application of1 mM aqueous solution (0.05 mL) including ICG or the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to a cotton swab (an upper photograph), and a state immediately after5-second washing with running water after 3 minutes (a lowerphotograph).

FIG. 3 shows results of adsorption tests of ICG and the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to a meat model of a living body. It shows a state immediately afterapplication of 1 mM aqueous solution (0.05 mL) including ICG or thecompound represented by the chemical formula 20 to a depression partwith a diameter of 5 mm of a pork loin meat (a left photograph), and astate immediately after 10-second washing with running water after 3minutes (a right photograph).

FIG. 4 shows results of adsorption tests of ICG and the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to a protein model of a living body. It shows a state immediately afterapplication of 1 mM aqueous solution (0.05 mL) including ICG or theisomerization equilibrium compound (TK1) represented by the chemicalformula 19 or 20 to a depression part with a diameter of 5 mm of achicken breast meat (a left photograph), and a state immediately after10-second washing with running water after 3 minutes (a rightphotograph).

FIG. 5 shows results of adsorption tests of ICG and the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20to a hydrophobic chemical fiber. It shows a state immediately afterapplication of 1 mM aqueous solution (0.05 mL) including ICG or theisomerization equilibrium compound (TK1) represented by the chemicalformula 19 or 20 to a polypropylene mask (a left photograph), and astate immediately after one-second washing with running water after 20minutes (a right photograph).

FIG. 6 shows results of molecule association tests of ICG and theisomerization equilibrium compound (TK1) represented by the chemicalformula 19 or 20. A left graph shows results of ICG, and a right graphshows results of the isomerization equilibrium compound (TK1)represented by the chemical formula 19 or 20.

FIG. 7 is a graph showing concentration dependence of a fluorescenceintensity in human venous blood of ICG, the isomerization equilibriumcompound (TK1) represented by the chemical formula 19 or 20, and theisomerization equilibrium compound (TK2) represented by the chemicalformula 15 or 16.

FIG. 8 is a view showing an observation state when ICG, isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20,or the isomerization equilibrium compound (TK2) represented by thechemical formula 15 or 16 is administered to a rat.

FIG. 9 shows views showing fluorescence states in an abdominal cavitywhen ICG, the isomerization equilibrium compound (TK1) represented bythe chemical formula 19 or 20, or the isomerization equilibrium compound(TK2) represented by the chemical formula 15 or 16 is administered to arat.

FIG. 10 shows views obtained by administrating a 0.1 mM aqueous solution(0.075 mL) including the isomerization equilibrium compound (TK2)represented by the chemical formula 15 or 16 to a caudate vein of a maleWistar rat (14-week old, 300 g), and taking an image thereof by using anear-infrared observation system PDE manufactured by Hamamatsu PhotonicsK.K. Left is a monochrome image through visible light, and right is afluorescence image.

FIG. 11 shows views obtained by administrating a 0.1 mM aqueous solution(0.075 mL) including the isomerization equilibrium compound (TK2)represented by the chemical formula 15 or 16 to a caudate vein of a maleWistar rat (14-week old, 300 g), and taking an image thereof by using anear-infrared fluorescent endoscope. Left is a monochrome image throughvisible light, and right is a fluorescence image.

FIG. 12 shows views of fluorescence states in a dorsal region of footwhen ICG, the isomerization equilibrium compound (TK1) represented bythe chemical formula 19 or 20 or the isomerization equilibrium compound(TK2) represented by the chemical formula 15 or 16 is administered to arat.

FIG. 13 is a graph showing changes with time in the fluorescenceintensity in a dorsal region of foot when ICG, the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20,or the isomerization equilibrium compound (TK2) represented by thechemical formula 15 or 16 is administered to a rat.

FIG. 14 is a graph showing changes with time in the fluorescenceintensity in a dorsal region of foot when ICG, the isomerizationequilibrium compound (TK1) represented by the chemical formula 19 or 20,or the isomerization equilibrium compound (TK2) represented by thechemical formula 15 or 16 is administered to a rat.

FIG. 15 is an illustration showing a procedure for directly observing ablood vessel of a rat.

FIG. 16 is a view showing an area (cremaster skin flap) directlyobserved in a blood vessel of a rat.

FIG. 17 is a view showing fluorescence states of a blood vessel of a ratto which ICG is administered and tissues around it.

FIG. 18 is a view showing fluorescence states of a blood vessel of a ratto which the isomerization equilibrium compound (TK1) represented by thechemical formula 19 or 20 is administered and tissues around it.

FIG. 19 is a graph showing volumes of a left foot of a rat measuredimmediately after an administration (post injection) and at one weekafter the administration (1 w after) in a group in which carrageeninadministration is performed (edema) and a group in which theadministration is not performed (normal) in experiment groups using TK1,wherein a vertical axis shows a volume (mL).

FIG. 20 is a graph showing results of Von Frey tests of a left foot of arat measured immediately after an administration (post injection) and atone week after the administration (1 w after) in a group in whichcarrageenin administration is performed (edema) and a group in which theadministration is not performed (normal) in experiment groups using TK1,wherein a vertical axis shows a load (g) when a response is obtained.

FIG. 21 is a graph showing changes in a luminance of a left foot surfaceafter TK1 was injected immediately after injection of carrageenin in agroup in which the carrageenin administration is performed (edema) and agroup in which the administration is not performed (normal), wherein avertical axis shows a luminance (arbitrary unit) and a horizontal axisshows a time (second).

FIG. 22 is a graph showing changes in a luminance of a left foot surfaceafter TK1 was injected one week after injection of carrageenin in agroup in which the carrageenin administration is performed (edema) and agroup in which the administration is not performed (normal), wherein avertical axis shows a luminance (arbitrary unit) and a horizontal axisshows a time (second).

FIG. 23 is a graph showing volumes of a left foot of a rat measuredimmediately after an administration (post injection) and at one weekafter the administration (1 w after) in a group in which carrageeninadministration is performed (edema) and a group in which theadministration is not performed (normal) in experiment groups using ICG,wherein a vertical axis shows a volume (mL).

FIG. 24 is a graph showing results of Von Frey tests of a left foot of arat measured immediately after an administration (post injection) and atone week after the administration (1 w after) in a group in whichcarrageenin administration is performed (edema) and a group in which theadministration is not performed (normal) in experiment groups using ICG,wherein a vertical axis shows a load (g) when a response is obtained.

FIG. 25 is a graph showing changes in a luminance of a left foot surfaceafter ICG was injected immediately after injection of carrageenin in agroup in which carrageenin administration is performed (edema) and agroup in which the administration is not performed (normal), wherein avertical axis shows a luminance (arbitrary unit) and a horizontal axisshows a time (second).

FIG. 26 is a graph showing changes in a luminance of a left foot surfaceafter ICG was injected one week after injection of carrageenin in agroup in which carrageenin administration is performed (edema) and agroup in which the administration is not performed (normal), wherein avertical axis shows a luminance (arbitrary unit) and a horizontal axisshows a time (second).

MODE FOR CARRYING OUT THE INVENTION

The diagnostic composition of the present invention can be used indiagnosis even if iodine is not included by employing thecyclodextrin-bonded indocyanine compound of the present inventiondescribed below as a pigment. The diagnostic composition can besubstituted for a diagnostic composition including an indocyanine greenwhich has hitherto been used. The application thereof may include, forexample, liver function test drugs, circulatory function test drugs, andthe like. In addition, the composition can be applied to medicaloperations and medial diagnosis in which near-infrared fluorescenceemitted by administration thereof to a body such as a blood vessel,lymph vessel, brain, eye, stomach, breast, esophagus, skin, kidney,ureter, bladder, urethra, lung, heart, or other moiety is observed. Itis considered that the pigment included in the diagnostic composition ofthe invention has a low binding property to a living body, and it canlabel a necessary moiety over a long time. The diagnostic compositionmay include a salt as an isotonizing agent and other additives ifnecessary. A form in which the ingredients are arbitrarily dissolved maybe employed, in addition to a form which is previously prepared in thestate of an aqueous solution. This diagnostic composition can beadministered by an injection, infusion, application or oraladministration.

This diagnostic composition can be preferably used when a circulation ofan aqueous solution such as blood, lymph fluid, interstitial tissuefluid, or urine is visualized. The visualization of the circulation ofblood can be used, for example, in a determination of necrosis byevaluating a peripheral circulation, an evaluation of tissue engagementafter a revascularization or transplant operation, a diagnosis of bloodcirculation failure, or the like. In addition, it is applicable to aneyeground imaging, an evaluation of a cerebral circulation, an imagingduring an operation in a brain surgical operation, an identification ofa sentinel lymph node in a cancer (a breast cancer, esophagus cancer,stomach cancer, colon cancer, prostate cancer, skin cancer, and thelike), an evaluation of lymphedema, an intraoperative cholangiography, amarking of tumor, a coronary artery imaging, an abdominal blood vesselimaging (hepatic artery, abdominal aorta, digestive tract blood flow,and the like), in these filed, the visualization has hitherto beenperformed. The composition also enables a fluorescence imaging of akidney and excretory system such as kidney, ureter, bladder, or urethra.

<1. Non-Inclusion Type Cyclodextrin-Bonded Indocyanine Compound andSynthesis Thereof>

The non-inclusion type cyclodextrin-bonded indocyanine compound of thepresent invention may include the chemical formula 1, the chemicalformula 3, the chemical formula 5, the chemical formula 7, the chemicalformula 9 and the chemical formula 11, and a synthesis method thereofcan be performed by reacting an indocyanine compound with a cyclodextrincompound in a solution.

In the chemical formulae 1 and 3, R₁ to R₄ and R₁₃ to R₁₆ desirably arenot bulky so that they do not hinder the inclusion by the cyclodextrin,considering the inclusion in the area corresponding to the cyclodextrin.Examples thereof are hydrogen, and alkyl groups and alkoxy groups havingabout 1 to 3 carbon atoms. The hydrogen, methyl group and methoxy groupare particularly desirable, and the hydrogen is more desirable. Also,R₅, R₆, R₁₁ and R₁₂ desirably are not bulky for the inclusion in thecyclodextrin, though not to the extent of R₁ to R₄ and R₁₃ to R₁₆.Examples thereof include hydrogen, and alkyl groups and alkoxy groupshaving about 1 to 6 carbon atoms. As R₁ to R₆ and R₁₁ to R₁₆ aremoieties included by the cyclodextrin, the moiety is desirablyhydrophobic as a whole, even when a hydrophilic functional group isintroduced thereto. As R₁₇, R₁₈, R₂₂ and R₂₃ have a little influence onthe inclusion in the cyclodextrin, they are not particularly limited solong as they are the substituents described above. R₇, R₁₀, R₁₉ and R₂₁are desirably hydrogen in terms of the easy synthesis. In addition, R₈,R₉ and R₂₀ are not particularly limited so long as they are thesubstituents described above.

When the non-inclusion type cyclodextrin-bonded indocyanine compound ofthe present invention is formed by covalently bonding the indocyaninecompound to the cyclodextrin compound through an amide bond, a synthesismethod of the non-inclusion type cyclodextrin-bonded indocyaninecompound can be performed by a dehydration-condensation reaction of theindocyaninecarboxylic acid compound with the aminocyclodextrin compoundin a solution.

<2. Inclusion Type Cyclodextrin-Bonded Indocyanine Compound>

The cyclodextrin-bonded indocyanine compound of the present invention isa cyclodextrin-bonded indocyanine compound represented by the chemicalformula 2 wherein an indocyanine is covalently bonded to a cyclic sugarchain cyclodextrin, which is characterized in that at least a part ofthe naphthyl group of the indocyanine is included in a cavity of thecyclodextrin. The compound may have a substituent on the indocyaninegroup so long as the naphthyl group of the indocyanine is included inthe cavity of the cyclodextrin, and the compound exhibits near-infraredfluorescence. Although various kinds of cyclodextrins are known, it isnecessary that the naphthyl group on the indocyanine is included in thecavity of the cyclodextrin. Examples thereof may include α-cyclodextrin,β-cyclodextrin and γ-cyclodextrin, preferably β-cyclodextrin. Thecyclodextrin may have a substituent.

In the chemical formulae 2 and 4, R₁ to R₄ and R₁₃ to R₁₆ desirably arenot bulky so that they do not hinder the inclusion in the cyclodextrin.Examples thereof are hydrogen, and alkyl groups and alkoxy groups havingabout 1 to 3 carbon atoms. The hydrogen, methyl group and methoxy groupare particularly desirable, and the hydrogen is more desirable. Also,R₅, R₆, R₁₁ and R₁₂ desirably are not bulky for the inclusion in thecyclodextrin, though not to the extent of R₁ to R₄ and R₁₃ to R₁₆.Examples thereof include hydrogen, and alkyl groups and alkoxy groupshaving about 1 to 6 carbon atoms. As R₁ to R₆ and R₁₁ to R₁₆ aremoieties included in the cyclodextrin, the moiety is desirablyhydrophobic as a whole, even when a hydrophilic functional group isintroduced thereto. As R₁₇, R₁₈, R₂₂ and R₂₃ have little influence onthe inclusion in the cyclodextrin, they are not particularly limited solong as they are the substituents described above. R₇, R₁₀, R₁₉ and R₂₁are desirably hydrogen in terms of the easy synthesis. In addition, R₈,R₉ and R₂₀ are not particularly limited so long as they are thesubstituents described above. It is enough that the bond between theindocyanine group and the cyclodextrin is a covalent bond, and it is notparticularly limited. Examples thereof may include an alkyl bond, aminobond, amide bond, double bond, triple bond, ester bond, ether bond, andthe like. If efficiency on the chemical synthesis is emphasized, theamide bond is preferred.

In order to include at least a part of the naphthyl group of theindocyanine in the cavity of the cyclodextrin, it is preferable to use aspacer and covalently bond the indocyanine to the cyclic sugar chaincyclodextrin through the spacer. At this time, when the length of thespacer in the chemical formula 2 is controlled, it is possible tocontrol a degree of the inclusion of the naphthyl group of theindocyanine in the cavity of the cyclodextrin.

The cyclodextrin-bonded indocyanine compound which is characterized inthat the indocyanine is covalently bonded to the cyclic sugar chaincyclodextrin through the spacer, and the naphthyl group of theindocyanine is included in the cavity of the cyclodextrin, accordingly,can be preferably exemplified by the compound represented by thechemical formula 4. In addition, preferable examples thereof arecompounds of the chemical formula 6, the chemical formula 8, or thechemical formula 10. Furthermore, the compound of the chemical formula12 is more preferable. With respect to m, n, p and q in the chemicalformula 6, m+p and n+q are each desirably 5 or more and 7 or less. Inevery chemical formula (the chemical formulae 1 to 10), the structure(spacer) between the nitrogen atom in the structure corresponding to theindocyanine and the oxygen atom in the structure corresponding to thecyclodextrin has desirably 7 or more and 9 or less atoms, consideringthe easy inclusion in the cyclodextrin.

<3. Synthesis of Inclusion Type Cyclodextrin-Bonded IndocyanineCompound>

The inclusion type cyclodextrin-bonded indocyanine compound of thepresent invention are compounds as described above, and the synthesismethod thereof can be performed by dissolving the non-inclusion typecyclodextrin-bonded indocyanine compound, which is synthesized as aboveand used as a synthesis precursor, in an aqueous solution. The aqueoussolution may include any material so long as it does not hinder theinclusion, and the water content is not particularly limited. Thesuitable temperature at the inclusion is from −20° C. to 100° C.,preferably from 0° C. to 50° C. The time required for the inclusion isabout one month immediately after the addition to the aqueous solution.As described above, it is clear that the inclusion reaction may takevarious formats depending on the property of the non-inclusion typecyclodextrin-bonded indocyanine compound, the inclusion reactiontemperature, the composition or the concentration of the aqueoussolution, and the like.

The non-inclusion type cyclodextrin-bonded indocyanine compound of thepresent invention can be converted into the inclusion typecyclodextrin-bonded indocyanine compound by dissolving it in a mediumincluding water. The water content in the medium is not particularlylimited, and it is desirably 50% by mass or more because when thecontent is higher, the inclusion easily advances in principle. If thereis a compound capable of forming the inclusion type cyclodextrin-bondedindocyanine compound in a medium other than the aqueous solution, it isnot necessary to perform the inclusion using the aqueous solution.

The inclusion type cyclodextrin-bonded indocyanine compound may beformed at the stage of the synthesis of the non-inclusion typecyclodextrin-bonded indocyanine compound, and in such a case theinclusion reaction is not required again.

<4. Synthesis Method of Non-Inclusion Type Cyclodextrin-BondedIndocyanine Compound (One Example of Synthesis)>

A synthesis of the compound represented by the chemical formula 11 istaken as an example. The indocyanine compound represented by thechemical formula 11 can be obtained, for example, by mixing the compoundrepresented by the chemical formula 13, which is synthesized in a methoddescribed in Non Patent Document 5, the compound represented by thechemical formula 14, which is synthesized in a method described in NonPatent Document 6, a dehydration-condensing agent such as awater-soluble carbodiimide (WSC: for example,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) ordicyclohexylcarbodiimide (DCC), and a medium such as pyridine,N,N-dimethylformamide or an aqueous solution; and reacting the mixtureat −20° C. to 60° C. for 10 minutes to 100 hours. In addition, anactivator such as 1-hydroxybenzotriazole (HOBt) may be added to activatethe reaction. The amount of the dehydration-condensing agent is 2-foldor more moles of that of the compound represented by the chemicalformula 13, and the amount of the medium used is not limited so long asthe reaction products are dissolved and the dehydration-condensationreaction is not hindered. The activator is not limited so long as itactivates the dehydration-condensation reaction, and the addition amountthereof is not limited so long as the dehydration-condensation reactionadvances as expected.

<5. Purification Method of Non-Inclusion Type Cyclodextrin-BondedIndocyanine Compound>

A mixture including the non-inclusion type cyclodextrin-bondedindocyanine compound synthesized in the method described above isdissolved in an acidic aqueous solution, the resulting solution isapplied to a reverse phase column chromatography, and elution thereof isperformed using, as an eluate, for example, any one of a mixed solutionof water and methanol including an acid, a mixed solution of water andacetonitrile including an acid, a mixed solution of water and ethanolincluding an acid, and a mixed solution of water and acetone includingan acid, whereby the non-inclusion type cyclodextrin-bonded indocyaninecompound can be isolated and purified in a high purity. The acid is notlimited so long as it does not decompose the non-inclusion typecyclodextrin-bonded indocyanine compound, the elution can be effectivelyperformed and a post-treatment can be easily performed after theelution. Examples thereof may include hydrochloric acid, trifluoroaceticacid, acetic acid, sulfuric acid, nitric acid, formic acid, and thelike. The hydrochloric acid, trifluoroacetic acid and acetic acid arepreferable, and the hydrochloric acid is more preferable. Theconcentration of the acid is not limited, so long as it does notdecompose the non-inclusion type cyclodextrin-bonded indocyaninecompound, the elution can be effectively performed and a post-treatmentcan be easily performed after the elution. It is preferably from 0.01 mMto 10 mM, more preferably from 0.1 mM to 1 mM. When the concentration isadjusted to this range, the desired compound is not decomposed, and itcan be quickly eluted. The medium is removed from the non-inclusion typecyclodextrin-bonded indocyanine compound eluted, whereby it can beobtained in a solid state. The medium can be removed by alyophilization.

<6. Synthesis Method (One Example of Synthesis) and Purification Method(One Example of Purification) of Inclusion Type Cyclodextrin-BondedIndocyanine Compound>

The non-inclusion type cyclodextrin-bonded indocyanine compoundrepresented by the chemical formula 11 which has been synthesized andpurified in the methods described above is the non-inclusion type, forexample, in DMSO, but it is turned into the inclusion type compoundrepresented by the chemical formula 12 immediately after it is dissolvedin water. This phenomenon can be confirmed by an ¹H NMR.

The inclusion type compound, accordingly, exists as the inclusion typecompound in an aqueous solution, and can be used as the inclusion typecompound. In addition, when water is removed from the aqueous solutionincluding the inclusion type compound, it is possible to obtain theinclusion type compound in the solid state.

<7. Device for Measuring Biokinetics and Device for VisualizingCirculation Applied to Living Body to which Diagnostic Composition isAdministered>

Device for Measuring Biokinetics

The present device is completed based on the feature in which thecyclodextrin-bonded indocyanine compound of the present inventionemployed as the diagnostic composition of the present invention(hereinafter may be arbitrarily referred to as the “compound of thepresent invention”) has different biokinetics such as a degree oftransfer into an interstitial tissue from that of ICG.

The compound of the present invention has difference from ICG in themetabolic rate and transfer speed from the circulatory system such asblood or lymph fluid into an interstitial tissue, and a proportion ofthe compound of the present invention which is uptaken into theinterstitial tissue is relatively higher than that of ICG. When thekinetics of the compound of the present invention in a living body areanalyzed, therefore, a mechanism of the body fluid shift in the livingbody (biokinetics) can be exactly evaluated. When the measurementconditions (an active state of a living body, an ambient temperature,and the like) are made stationary as much as possible upon the use ofthe present device, the biokinetics can be more exactly measured.

One form of an abnormal body fluid shift in a living body is generationof edema. Here, according to so-called “Starling hypothesis,” it isgenerally considered that a body fluid (fluid) in a living body flowsfrom an artery to an interstitial tissue, and then from the interstitialtissue to a vein.

It can be considered that this fluid shift from the artery to the veincan be calculated as (A)−(B)−(C)+(D) wherein (A) is a fluid shiftflowing toward the outside of a circulatory system caused by adifference between an artery pressure and an interstitial tissuepressure, (B) is a fluid shift flowing toward the inside of thecirculatory system caused by a difference between a vein pressure andthe interstitial tissue pressure, (C) is a fluid shift flowing from aninterstitial tissue into an artery caused by a difference between anosmotic pressure of interstitial tissue fluid and an osmotic pressure ofarterial blood, and (D) is a fluid shift flowing from the interstitialtissue into a vein caused by a difference between an osmotic pressure ofthe interstitial tissue fluid and an osmotic pressure of venous blood.

When the difference between the artery pressure and the vein pressurebecomes smaller due to heart failure, or the osmotic pressure of theinterstitial tissue fluid lowers due to malnutrition, accordingly, thebalance of the fluid shifts (A) to (D) breaks, thus resulting ingeneration of edema. In addition, when permeability of a capillaryvessel becomes abnormal (abnormal selectivity of material penetration)as in diabetes mellitus, the balance of the fluid shifts (A) to (D) alsobreaks, thus resulting in generation of edema. Here, there has been acondition in which there is no simple method for directly evaluating anabnormal permeability of a capillary vessel, and therefore it hashitherto been difficult to specify a detail cause of generation of edemaeven if the generation of edema, which is a phenomenon, can be detected.

Under the finding described above, it can be expected that transfer ofthe compound of the present invention is changed by reduction of adifference between an artery pressure and a vein pressure or reductionof an osmotic pressure in a cell, compared to a case in which edema isgenerated, when material penetration becomes abnormal at a blood vesselwall of a capillary vessel. When the transfer of the compound of thepresent invention is observed in an amount equal to or more than (orless than) that corresponding to the fluid shift, accordingly, thegeneration of abnormal permeability of the capillary vessel can bedetermined. It can be considered that when the permeability of thecapillary vessel does not become abnormal, the change of the transfer ofthe compound of the present invention corresponds to an extent of thefluid shift, and therefore it can be distinguished from the case inwhich the blood vessel wall becomes abnormal.

In particular, when two or more kinds of the compounds of the presentinvention having a transfer speed into the interstitial tissue differentfrom each other are employed, and the biokinetics of these compounds aremeasured and compared with each other, the degree of change in thepermeability of the capillary vessel may possibly be evaluated; in otherwords, when two or more kinds of the compounds of the present inventionhaving a different permeability from each other through the blood vesselwall of the capillary vessel are employed, and the permeabilitiesthereof are measured, it becomes possible to more exactly evaluatewhether or not the permeability becomes abnormal at the blood vesselwall. This means that the cause of the change in the fluid shift can bemore exactly evaluated by employing two or more kinds of the compoundsof the present invention having a different sensitivity to the abnormalpermeability of the blood vessel wall to each other, and measuring thebiokinetics thereof. In addition to the evaluation of the fluid shift byusing the compound of the present invention, the fluid shift can bemeasured in a usual procedure.

The device for measuring biokinetics of the present invention hasexcitation light-irradiating means, fluorescence intensity-measuringmeans, biokinetics-calculating means, and other means. The other meansis selected as occasion demand, and example thereof is concentration ina living body-calculating means of calculating a concentration of thecyclodextrin-bonded indocyanine compound from the fluorescence intensitymeasured by the fluorescence intensity-measuring means.

The present device is a device used for measuring at least a part of aliving body to which the diagnostic composition of the present inventiondescribed above is administered. The amount of the diagnosticcomposition administered is an amount at which fluorescence can beobserved when excitation light is irradiated to the moiety to bemeasured. The appropriate amount, accordingly, varies depending on themeasurement moiety. The kind of the compound of the present inventionincluded in the diagnostic composition is not particularly limited. Thecompounds of the present invention may be used alone or as a mixture oftwo or more kinds. A diagnostic composition having the same or differentcomposition may be additionally administered in the course of themeasurement.

The excitation light-irradiating means is means of irradiatingexcitation light having a wavelength capable of generating fluorescencefrom the compound of the present invention included in the diagnosticcomposition administered. The wavelength of the excitation lightirradiated can be restricted to an appropriate range. When thewavelength is restricted to a range as narrow as possible, thefluorescence can be certainly separated from the excitation light. Thewavelength can be restricted by selecting a light source capable ofemitting light having an appropriate wavelength, or restricting awavelength through a filter.

The mode of irradiation of the excitation light is not particularlylimited so long as the fluorescence generated can be measured by thefluorescence intensity-measuring means described below. Example thereofmay include contiguous excitation lights, pulsatile excitation light,excitation light whose intensity is variable, and the like. When theintensity is changed, the intensity of the excitation light can bemodulated by irradiating pulses of the excitation light in apredetermined interval, and the like. It is desirable to modulate theintensity of the excitation light by employing pulse-amplitudemodulation.

The excitation light is irradiated to a moiety to be irradiated by anappropriate optical system. The moiety to be irradiated is a moietywhich requires measurement of biokinetics, for example, when anevaluation of a moiety in which edema is generated is intended, it isdesirable to directly irradiate the excitation light to the moiety wherethe edema is generated.

A range to which the excitation light is irradiated is not particularlylimited. The irradiation range is determined according to the need. Whenthe light is irradiated to a narrow range, subdivided biokinetics can beprecisely measured in the narrow range irradiated. When the light isirradiated to a wide range, a relative amount of the compound of thepresent invention which emits the fluorescence by the irradiation of theexcitation light is increased, and thus the fluorescence intensity canbe more precisely measured.

Further, it is preferable to irradiate the excitation light by theexcitation light-irradiating means under a condition in which influenceof ambient light is suppressed. For example, it is preferable toirradiate the excitation light in a dark place, or in a condition inwhich an area to which the excitation light is irradiated is coveredfrom light from outside.

The fluorescence intensity-measuring means is means of measuring anintensity of the fluorescence emitted from a moiety in which theexcitation light is irradiated by the excitation light-irradiatingmeans. It is preferable to measure light from which light other than thefluorescence (ambient light, excitation light, and the like) is removedthrough a filter capable of selectively passing the fluorescenceemitted.

When means of irradiating excitation light having a modulated intensityis employed as the excitation light-irradiating means, a componentshowing a change corresponding to the modulation can be separated fromlight intensity measured, which can be used as the fluorescenceintensity. For example, when the intensity of the excitation light ismodulated by pulse-amplitude modulation, a component of light whichchanges depending on the intensity of the pulse modulated isdemodulated, and its intensity is measured, thereby separating thefluorescence intensity. This can reduce the influence by ambient lighton the measurement results of the fluorescence intensity.

The concentration in a living body-calculating means is means ofcalculating a concentration of the compound of the present invention inthe living body based on the fluorescence intensity measured by thefluorescence intensity-measuring means. The relationship between thefluorescence intensity and the concentration of the compound of thepresent invention in the living body can be calculated in an appropriatemethod. For example, a calibration curve is previously made, and aconcentration of the compound of the present invention in a living bodycan be calculated based on the calibration curve. The absolute value ofthe fluorescence intensity can also be used as it is as a value relatingto a concentration of the compound of the present invention in a livingbody. The concentration in a living body-calculating means calculateswith time a concentration of the compound of the present invention in aliving body.

The biokinetics-calculating means is means of obtaining a time-rate ofchange in the concentration of the compound of the present invention ina living body from the data of the concentrations in the living bodyacquired with time by the concentration in a living body-calculatingmeans. The biokinetics-calculating means calculates a transfer speed ofthe compound of the present invention from the circulatory system intothe interstitial tissue or from the interstitial tissue into thecirculatory system in the part of the living body, from the resultingtime-rate of change in the concentration in the living body. Thetransfer speed into or out of the interstitial tissue may also directlybe calculated from the change in the fluorescence intensity obtainedwith time by the fluorescence intensity-measuring means, without usingthe concentrations in the living body.

Here, it can be assumed that there is a high correlation between thetransfer of the compound of the present invention toward the inside(outside) of the interstitial tissue and the fluid shift in the bodyfluid, and therefore a state of fluid shift in a measurement moiety canbe evaluated by measuring biokinetics of the compound of the presentinvention. In addition, it can be considered that when the blood vesselwall becomes abnormal and the material permeability is not normal, thekinetics of the compound of the present invention are changed in aliving body. The permeability of the blood vessel, therefore, can beevaluated by the evaluation of the kinetics of the compound of thepresent invention.

The time-rate of change in the fluorescence intensity (or theconcentration in the living body) can be obtained by differentiating thechange with time in the fluorescence intensity (or the concentration inthe living body) with time, or by calculating a fluorescence intensity(a concentration in a living body) per predetermined time to obtain adifference from a fluorescence intensity (or a concentration in a livingbody) before the predetermined time (after the predetermined time).

An uptake or excretion speed into or out of the interstitial tissuefluid of the compound of the present invention in a part of the livingbody is calculated, from the resulting time-rate of changes in thefluorescence intensity (or the concentration in the living body). Here,the uptake or excretion speed into or out of the interstitial tissuefluid can be obtained as a relative value from the fluorescenceintensity, and also it can be calculated based on the concentration ofthe compound of the present invention in the interstitial tissue and theamount of the interstitial tissue fluid, when it is desired to obtain itmore precisely. In addition, when the concentration and the amount ofthe compound of the present invention in blood or lymph fluid are takeninto account, more precise calculation can be performed. Theconcentration of the compound of the present invention in blood or thelike can be precisely measured by actually sampling the blood.

The method for calculating a concentration in the interstitial tissuefluid may be exemplified by the following methods. A first method is amethod in which the concentration of the compound of the presentinvention in the living body calculated is approximated as theconcentration thereof in interstitial tissue fluid in a part of a livingbody as it is. The concentration in the interstitial tissue fluid can becalculated in consideration of the amount of the interstitial tissuefluid as a method relevant to the first method. A second method is amethod in which a concentration of the compound of the present inventionin blood is actually measured, a proportion of contribution by materialsin the blood vessel to a value measured by the fluorescenceintensity-measuring means is calculated by a method using anotherstandard substance which does not transfer to the interstitial tissue(such as ICG), and a concentration in the interstitial tissue fluid canbe calculated by subtracting the influence caused by the concentrationof the compound of the present invention in blood actually measured.

The time-rate of change in the concentration of the compound of thepresent invention in the interstitial tissue fluid can be calculated bysubtracting the transfer speed out of the interstitial tissue from thetransfer speed into the interstitial tissue. Here, when it is assumedthat the transfer of the compound of the present invention into (or outof) the interstitial tissue advances in a speed correlating to a shiftspeed of fluid in body fluid and the fluid shift reaches equilibrium,the transfer speed into the interstitial tissue and the transfer speedout of the interstitial tissue can be considered as constants. Thetransfer speeds, therefore, can be calculated from the change in theconcentration of the compound of the present invention in blood (thechange caused by metabolism, excretion, transfer to tissues, and thelike), and the time-rate of change in the concentration in theinterstitial tissue fluid. Even if these transfer speeds vary, thetransfer speeds can be calculated by making a model corresponding to thechange and applying thereto.

When the transfer speed calculated is out of a range of values shown bya normal blood vessel wall, it can be considered that the blood vesselwall may become abnormal. There can be a case in which the permeabilityof the compound of the present invention is increased as well as a casein which it is decreased resulting from the abnormality of the bloodvessel wall.

It is a well-known fact that tissue becomes acutely tumescent due tovarious tissue injuries such as acute inflammation, ischemia or trauma,and this is a very important tissue reaction in the medical setting,regardless of the region.

It is an item relevant to whole organs, for example, in an evaluation ofsuture of an intestine during an operation, an evaluation of a degree ofan inflammation such as pneumonia, an evaluation of compatibility in anorgan transplant, an evaluation of dysfunction in brain, kidney, and thelike, in addition to the case in which the generation of tumescenceitself becomes problems.

However, there is no means of exactly predicting a possibility or adegree of tissue tumescence at the current moment, and it is onlyempirically predicted from a degree of a tissue injury. In most cases,accordingly, a treatment must be considered after the generation oftumescence is confirmed, which is a big therapeutic restrictions.

If the possibility of tumescence generated can be early predicted in ahigh precision, a treatment of reducing the tumescence can be earlyperformed whereby the influence can be suppressed to the minimum. Forexample, if a degree of a brain edema, which will be caused later, canbe exactly predicted immediately after bleeding from the brain, theinfluence can be kept to the minimum by early performing a decompressiontreatment or a treatment for reducing a blood vessel permeability.Similarly, if an influence caused by ischemia stress can be predicted inadvance, a secondary damage caused by myocardial infarction or extremityinjury can be kept to the minimum. As stated above, a technique ofhighly precision, quantitative prediction of tumescence has a potentialto provide considerable impact on the entirety of the medical practice.

The quantification of tumescence of tissues including edema has hithertobeen qualitatively evaluated from an appearance. The quantification hasbeen tried, but it can be performed only under very limited conditions,and has a defect in which the evaluation can be performed only after thegeneration of the tumescence.

In order to solve these problems, when the device for measuringbiokinetics of the present invention is used, the tumescence progressioncan be predicted by calculating a fluid shift speed into or out of theinterstitial tissue. As described in detail in Examples, it is knownthat ICG does not transfer to the interstitial tissue during thetumescence progression. On the other hand, it is known that thecyclodextrin-bonded indocyanine compound of the present invention cantransfer into or out of the interstitial tissue, and the transfer intothe interstitial tissue is further promoted with the fluid shift duringthe tumescence progression. The tumescence progression, accordingly, canbe predicted by evaluating the transfer of the cyclodextrin-bondedindocyanine compound of the present invention into the interstitialtissue.

As it is considered that the fluid shift speed into or out of theinterstitial tissue exerts influence on how big tumescence finallyformed is, evaluation of the transfer speed of the cyclodextrin-bondedindocyanine compound of the present invention, which is relevant to thefluid shift speed, can leads to the evaluation of a degree of finaltumescence progression. The transfer speed can be obtained bycalculating a concentration of the cyclodextrin-bonded indocyaninecompound, or it may be calculated by using the fluorescence intensity asit is. The transfer speed into or out of the interstitial tissue can becalculated as an absolute value needless to say, and also a relativevalue (for example, a rate of change in the fluorescence intensity maybe adopted as a value relevant to the transfer speed as it is) iscalculated and tumescence progression can be predicted according to therelative value.

The calculation of the tumescence progression is desirably performedbased on a transfer speed into the interstitial tissue in a normalperiod in which the tumescence does not advance. There are, however,cases in which the transfer speed into the interstitial tissue is notfound in the normal period, and in such cases, therefore, a moiety inwhich the tumescence does not advance is selected as a control moiety,and a transfer speed obtained in that moiety can be used insteadthereof. The degree of the transfer of the cyclodextrin-bondedindocyanine compound of the present invention into the interstitialtissue may also be evaluated by evaluating an amount of bloodcirculating and a degree of metabolism using ICG which does not transferinto the interstitial tissue.

The degree of transfer of the cyclodextrin-bonded indocyanine compoundof the present invention into the interstitial tissue is measured aftera predetermined time course from the administration of the diagnosticcomposition of the present invention to a living body. The predeterminedtime refers to a time necessary for distribution of the diagnosticcomposition of the present invention in blood. The reason why theevaluation is performed using data after the predetermined time courseis that there is almost no difference in the change of fluorescenceintensity regardless of the tumescence progression, because theconcentration in blood is quickly elevated with almost no influencecaused by a degree of tumescence progression until the diagnosticcomposition is distributed in blood over the predetermined time afterthe administration thereof. Once it is distributed in blood, thetransfer speed into the interstitial tissue is changed depending on thepresence or absence of the tumescence progression, and accordingly itcan be observed selective increase of the fluorescence intensity in amoiety in which the transfer advances (i.e., a moiety in which thetumescence advances).

A concrete method for presuming the tumescence progression may include,for example, a method in which a blood volume is presumed from a bodyweight or the like, a size of a peak of the fluorescence intensity and atime reaching the peak are calculated from the blood volume presumed.Considering the time and the intensity, a fluorescence intensity ismeasured at a time when the intensity will be changed with generation ofedema, and a degree of edema progression and a degree of edema whichwill be probably generated in future are predicted from the obtainedresults.

Device for Visualizing Circulation

The device is completed based on the fact in which thecyclodextrin-bonded indocyanine compound of the invention (hereinaftermay be arbitrarily refereed to as the “compound of the presentinvention”) employed in the diagnostic composition of the invention isdifferent from ICG in biokinetics such as a degree of transfer into aninterstitial tissue.

The compound of the present invention easily transfers into interstitialtissue fluid, and thus it becomes possible to visualize a circulation ofblood or lymph fluid by tracing the behavior of the compound of thepresent invention.

The device for visualizing circulation of the present invention hasexcitation light-irradiating means, fluorescence-imaging means,morphological imaging means and displaying means. The device is a deviceused for measuring at least a part of the living body to which thediagnostic composition of the invention described above is administered.The amount of the diagnostic composition administered is an amount atwhich fluorescence can be observed when excitation light is irradiatedto the moiety to be measured. The appropriate amount, accordingly,varies depending on the measurement moiety. The kind of the compound ofthe present invention included in the diagnostic composition is notparticularly limited. The compounds of the present invention may be usedalone or as a mixture of the two or more kinds. A diagnostic compositionhaving the same or different composition may be additionallyadministered in the course of the measurement.

The excitation light-irradiating means is means of irradiatingexcitation light having a wavelength capable of generating fluorescencefrom the compound of the present invention included in the diagnosticcomposition administered. The wavelength of the excitation lightirradiated can be restricted to an appropriate range. When thewavelength is restricted to a range as narrow as possible, thefluorescence can be certainly separated from the excitation light. Thewavelength can be restricted by selecting a light source capable ofemitting light having an appropriate wavelength, or restricting awavelength through a filter.

The mode of irradiation of the excitation light is not particularlylimited so long as the fluorescence generated can be measured by thefluorescence-imaging means described below. Example thereof may includecontiguous excitation lights, pulsatile excitation light, excitationlight whose intensity is variable, and the like. When the intensity ischanged, the intensity of the excitation light can be modulated byirradiating pulses of the excitation light in a predetermined interval,and the like. It is desirable to modulate the intensity of theexcitation light by employing pulse-amplitude modulation.

The excitation light is irradiated to a moiety to be irradiated by anappropriate optical system. The moiety to be irradiated is a moiety inwhich the visualization of circulation is required in a living body, andmay include, for example, a moiety at which necrosis of tissue isadvancing due to thermal injury, frostbite, inflammation, wound orinfarction, and periphery thereof. As Blood is not circulated in themoiety in which the tissue is necrotized and there is little advantageeven if that moiety is left as it is, the removal thereof is thought. Insuch a case, it is ideal to completely remove the necrosis area aloneamong the normal area and the necrosis area.

The identification of the necrosis area has hitherto been performed by ablood vessel angiography in which a contrast agent is administered, or amethod in which hypothermia is detected with decrease of bloodcirculation. The blood vessel angiography, however, has a defect inwhich it is not easy to handle devices such as X-ray irradiation device,and according to the evaluation method based on the body temperature, itis not easy to obtain an exact determination.

The device of the present invention can be used for an application inwhich evaluation of the necrosis area and the normal area is performeddue to presence or absence of the circulation of body fluid (blood). Ifa moiety with no circulation can be visualized, then the moiety can beeasily removed. In addition to the visualization of the necrosis area,it becomes possible to easily evaluate occurrence of abnormality in thecirculatory function, because the blood circulation can be directlyobserved. For example, a moiety in which necrosis does not occur yet,but a circulatory function is decreased due to infarction can bevisualized.

The range to be irradiated by the excitation light is decided so as toinclude an area requiring the visualization of the circulation asoccasion demands.

Further, it is preferable to irradiate the excitation light by theexcitation light-irradiating means under a condition in which influenceof ambient light is suppressed. For example, it is preferable toirradiate the excitation light in a dark place, or in a condition inwhich an area to which the excitation light is irradiated is coveredfrom light from outside.

The fluorescence-imaging means is a means of two-dimensionally acquiringan intensity of fluorescence emitted by the compound of the presentinvention which has been excited by the excitation light-irradiatingmeans to obtain distribution state data of the compound of the presentinvention in a living body; i.e., this means is means of acquiring thedistribution state data showing the distribution of the compound of thepresent invention in a part of a living body as two-dimensional imagedata.

It may be formed of, for example, a combination of an appropriateoptical system and an image pick-up device such as CCD. A resolution ofthe data two-dimensionally acquired is set to a necessary valuedepending on the purpose. It is preferable to measure the fluorescenceintensity of light from which light other than the fluorescence (ambientlight, excitation light, and the like) is removed through a filtercapable of selectively passing the fluorescence emitted.

When means of irradiating excitation light having a modulated intensityis employed as the excitation light-irradiating means, a componentshowing a change corresponding to the modulation can be separated fromlight intensity measured, which can be used as the fluorescenceintensity. For example, when the intensity of the excitation light ismodulated by pulse-amplitude modulation, a component of light whichchanges depending on the intensity of the pulse modulated isdemodulated, and its intensity is measured, thereby separating thefluorescence intensity. This can reduce the influence by ambient lighton the measurement results of the fluorescence intensity.

The morphological imaging means is means of two-dimensionally acquiringan intensity of light having a wavelength other than the wavelength ofthe fluorescence emitted by the compound of the present invention toobtain morphological data of a part of a living body, i.e., the means ismeans of acquiring morphological data showing the morphology of the partof the living body as two-dimensional image data.

The morphological imaging means can be formed of an appropriate opticalsystem and an image pick-up device such as CCD. A resolution of the datatwo-dimensionally acquired is set to a necessary value depending on thepurpose. In such a case, it is set so that the fluorescence emitted fromthe excitation light is not detected (or the detection sensitivity islowered). As the morphological imaging means, a structure in which mostof the optical system is used for this means and the fluorescenceimaging means described above, and the light having a wavelengthcorresponding to the fluorescence is introduced into thefluorescence-imaging means and lights having other wavelengths isintroduced in to the morphological imaging means through a spectralprism in an optical path before final introduction into the imagepick-up device can be adopted. The spectral prism can appropriatelycontrol the wavelength of light to be separated by appropriately forminga dichroic film.

Further, one imaging device can be used as the fluorescence-imagingmeans and as the morphological-imaging means. The fluorescence may bemathematically separated from other lights after two-dimensional imagedata are obtained. Furthermore, the distribution state data can also beobtained as multiple image data from a surface of apart of a living bodyin a depth direction. A focal point in the optical system adopted in thefluorescence-imaging means is shifted in the depth direction, wherebymultiple two-dimensional image data can be obtained in a depthdirection. In addition, an optical system capable of changing a focallength is adopted as the excitation light-irradiating means, and thefocal point is shifted not only on the surface of the part of the livingbody but also in the depth direction, or excitation light thinly stoppedis irradiated to the part of the living body, whereby the fluorescencecan be selectively generated not only on the surface of the living bodybut also on the inside in the depth direction of the living body, andthe circulation at that area can be visualized.

The displaying means is means of displaying a distribution state of thecompound of the present invention in a part of a living body byoverlapping the distribution state data obtained by thefluorescence-imaging means with the morphological data obtained by themorphological imaging means. When a wavelength of fluorescence is notwithin a range of visible light, the wavelength of fluorescence isconverted to an appropriated wavelength of visible light, which isdisplayed. It is desirable to display the distribution state data inpreference to the morphological data in terms of the visualization ofthe circulation. In particular, an area in which a distribution quantity(i.e., a fluorescence intensity) of the compound of the presentinvention is low (whether it is low or not is decided in accordance withthe purpose of visualizing the circulation. For example, for the purposeof visualizing a necrosis area, it is an area in which the circulationis not observed, i.e., an area in which fluorescence is not observed) isdisplayed so that it can be distinguished from other area. For example,the area can be displayed by a color different from that in anotherarea, or can be displayed by blinking it. The overlapping of thedistribution state data with the morphological data can be realized bylogic on a computer. The two-dimensional data can be displayed by usinga usual display device. When the display device is placed between aliving body and a measurer, a living body can be treated while lookingat the display device.

<8. Definitions, and the Like>

In the present invention, “alkyl group” refers to a linear or branchedalkyl group having 1 to 20 carbon atoms, which may have a substituent,and examples thereof may include linear groups or branched groupsincluding methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,hexadecyl, heptadecyl, octadecyl, nonadecyl and icosanyl.

In the present invention, “alkoxyl group” may include linear or branchedalkoxyl group having 1 to 20 carbon atoms, such as methoxy, ethoxy,propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy,ethoxyethoxy, ethoxypropoxy and methoxyethoxyethoxy groups.

In the present invention, “aryl group” may include aromatic hydrocarbonshaving 6 to 20 carbon atoms, such as phenyl and naphthyl.

EXAMPLE

The preferable embodiments of the present invention will be specificallyexplained by means of Examples, but the technical scope of the presentinvention is not limited to embodiments described below, and variousmodifications thereof can be carried out in the scope of the presentinvention.

<Test 1: Synthesis and Purification of Compounds Represented by ChemicalFormula 15 and Chemical Formula 16>

A mixture of 0.20 g of compound represented by the chemical formula 13,0.94 g of a compound represented by the chemical formula 14, 0.18 g ofWSC, 0.12 g of HOBt, 4.0 mL of pyridine and 2.0 mL ofN,N-dimethylformamide was stirred at 0° C. for 6 hours in a dark place.After that, 50 mL of acetone was added thereto, a precipitate wasfiltered under a reduced pressure. The precipitate was dissolved in anaqueous solution of 0.1% trifluoroacetic acid, and the solution wassubjected to an ODS column chromatography. A mixed liquid of water andmethanol including 1 mM hydrochloric acid was used as an eluate, and acompound represented by the chemical formula 15 was eluted. The eluateis concentrated under a reduced pressure to obtain 0.65 g of a greeninclusion type compound represented by the chemical formula 16 in asolid state (In the concentration of the eluate under reduced pressure,the product is naturally turned into an inclusion type, because a watercontent is high at the end of the concentration).

Mechanical analysis data of the desired product represented by thechemical formula 16 are shown below. ¹H NMR (500 MHz, D₂O, 26° C.,Acetone: 2.10 ppm) 1.43 (2H, m), 1.55 (2H, m), 1.99 (6H, s), 2.09 (6H,s), 2.63 (6H, m), 2.80 (6H, m), 2.92 (2H, m), 3.02 (2H, dd, J=3.7, 9.8Hz), 3.08 (2H, t, J=9.2 Hz), 3.2-4.1 (m), 4.19 (2H, t, J=9.8 Hz), 4.26(2H, t, J=9.8 Hz), 4.33 (2H, m), 4.43 (2H, m), 4.71 (2H, d, J=2.4 Hz),4.81 (4H, d, J=3.7 Hz), 4.91 (2H, d, J=3.7 Hz), 4.99 (2H, d, J=3.7 Hz),5.08 (2H, d, J=3.7 Hz), 5.13 (2H, d, J=3.1 Hz), 6.15 (2H, d, J=13 Hz),6.52 (2H, t, J=12 Hz), 7.43 (4H, m), 7.57 (1H, d, J=12 Hz), 7.57 (2H, d,J=9.2 Hz), 7.78 (2H, m), 8.06 (3H, m), 8.15 (2H, d, J=8.5 Hz). ESI-MSm/z calcd for C₁₃₁H₁₉₁N₄O₇₂2972, found 2973 [M]⁺.

<Test 2: Synthesis and Purification of Compounds Represented by ChemicalFormula 19 and Chemical Formula 20>

A mixture of 0.17 g of a compound represented by the chemical formula17, 5 mL of methanol, and 0.30 g of t-BuOK was stirred at roomtemperature for 12 hours. After that, 3 mL of 1 M of hydrochloric acid,and then 50 mL of water were added thereto. A precipitate was filtered,washed with water, and dried under a reduced pressure to obtain 0.17 gof a compound represented by the chemical formula 18.

A mixture of 0.02 g of the compound represented by the chemical formula18, 0.081 g of a compound represented by the chemical formula 14, 0.016g of WSC, 0.011 g of HOBt, 0.3 mL of pyridine and 0.2 mL ofN,N-dimethylformamide was stirred at 0° C. for 6 hours in a dark place.After that, 5 mL of acetone was added thereto, and a precipitate wasfiltered under a reduced pressure. The precipitate was dissolved in anaqueous solution of 0.1% trifluoroacetic acid, and the solution wassubjected to an ODS column chromatography. A mixed liquid of water andmethanol including 1 mM hydrochloric acid was used as an eluate, and acompound represented by the chemical formula 19 was eluted. The eluatewas concentrated under a reduced pressure to obtain 0.045 g of a greeninclusion type compound represented by the chemical formula 20 in asolid state. (In the concentration of the eluate under a reducedpressure, the product is naturally turned into an inclusion type,because a water content is high at the end of the concentration.)

Mechanical analysis data of the desired product represented by thechemical formula 20 are shown below. ¹H NMR (500 MHz, D₂O, 40° C.,Acetone: 2.26 ppm) 1.54 (2H, m), 1.68 (2H, m), 1.98 (2H, m), 2.19 (6H,s), 2.20 (2H, m), 2.30 (6H, s), 2.6-2.85 (10H, m), 2.95 (2H, m), 3.00(4H, m), 3.08 (2H, t, J=12 Hz), 3.17 (2H, dd, J=3.7, 9.8 Hz), 3.26 (2H,t, J=9.8 Hz), 3.35-4.30 (m), 4.35 (2H, t, J=9.2 Hz), 4.50 (2H, t, J=9.2H), 4.52 (2H, m), 4.63 (2H, m), 4.87 (2H, d, J=3.7 Hz), 4.95 (d, J=3.1Hz), 4.97 (2H, d, J=3.7 Hz), 5.08 (2H, d, J=3.7 Hz), 5.15 (2H, d, J=4.3Hz), 5.25 (2H, d, J=3.7 Hz), 5.29 (2H, d, J=3.7 Hz), 6.30 (2H, d, J=14.6Hz), 7.58 (4H, m), 7.73 (2H, d, J=8.5 Hz), 7.95 (2H, m), 8.25 (2H, m),8.32 (2H, d, J=14.6 Hz), 8.35 (2H, d, J=8.5 Hz). ESI-MS m/z calcd forC₁₃₅H₁₉₇N₄O₇₃ 3042, found 3042 [M]⁺.

<Test 3: Synthesis and Purification of Compound Represented by ChemicalFormula 21>

A mixture of 0.04 g of a compound represented by the chemical formula13, 0.18 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.05 g of WSC, 0.025g of HOBt, 0.8 mL of pyridine and 0.4 mL of N,N-dimethylformamide wasstirred at 0° C. for 3 hours in a dark place. After that, 10 mL ofacetone was added thereto, and a precipitate was filtered under areduced pressure. The precipitate was dissolved in an aqueous solutionof 0.1% trifluoroacetic acid, and the solution was subjected to an ODScolumn chromatography. A mixed liquid of water and methanol including 1mM hydrochloric acid was used as an eluate, and a compound representedby the chemical formula 21 was eluted. The eluate was concentrated undera reduced pressure to obtain 0.11 g of the green compound represented bythe chemical formula 21 in a solid state.

Mechanical analysis data of the desired product represented by thechemical formula 21 are shown below. ¹H NMR (500 MHz, D₂O, 29° C.,Acetone: 2.10 ppm) 1.79 (12H, br.), 2.68 (4H, br.), 3.0-4.5 (98H), 4.4(4H, br.), 4.5-5.3 (14H, br.), 6.18 (2H, br.), 6.46 (2H, br.), 7.3-8.2(15H). ESI-MS m/z calcd for C₁₂₅H₁₇₉N₄O₇₀ 2856, found 2856 [M]⁺.

<Test 4: Synthesis and Purification of Compound Represented by ChemicalFormula 23>

A mixture of 0.02 g of a compound represented by the chemical formula22, 0.096 g of mono-6-amino-6-deoxy-β-cyclodextrin, 0.032 g of WSC, 0.5mL of pyridine and 0.05 mL of 0.1 M phosphoric acid buffer was stirredat room temperature for 24 hours in a dark place. After that, 10 mL ofacetone was added thereto, and a precipitate was filtered under areduced pressure. The precipitate was dissolved in an aqueous solutionof 0.1% trifluoroacetic acid, and the solution was subjected to an ODScolumn chromatography. A compound represented by the chemical formula 23was eluted from the eluate using a mixed liquid of water andacetonitrile. The eluate was concentrated under a reduced pressure toobtain 0.014 g of the green compound represented by the chemical formula23 in a solid state.

Mechanical analysis data of the desired product represented by thechemical formula 23 are shown below. ¹H NMR (500 MHz, D₂O, 26° C.,Acetone: 2.15 ppm) 1.26 (4H, m), 1.5-2.25 (24H, br), 2.7-4.2 (88H), 4.82(2H, br), 4.90 (8H, br), 4.97 (2H, br), 5.03 (2H, br), 6.11 (2H, br),6.36 (2H, br), 7.3-8.01 (15H, br). ESI-MS m/z calcd for C₁₃₁H₁₉₁N₄O₇₀2940, found 2940 [M]⁺.

<Test 5: Synthesis and Purification of Compound Represented by ChemicalFormula 24>

A mixture of 0.20 g of a compound represented by the chemical formula22, 0.02 g of 3-amino-3-deoxy-β-cyclodextrin, 0.096 g of WSC, 0.013 g ofHOBt, 0.4 mL of pyridine and 0.2 mL of N,N-dimethylformamide was stirredat room temperature for one hour in a dark place. After that, 10 mL ofacetone was added thereto, and a precipitate was filtered under areduced pressure. The precipitate was dissolved in an aqueous solutionof 0.1% trifluoroacetic acid, and the solution was subjected to an ODScolumn chromatography. A compound represented by the chemical formula 24was eluted from the eluate using a mixed liquid of water and methanol.The eluate was concentrated under a reduced pressure to obtain 0.013 gof the green compound represented by the chemical formula 24 in a solidstate.

Mechanical analysis data of the desired product represented by thechemical formula 24 are shown below. ¹H NMR (500 MHz, D₂O, 29° C.,Acetone: 2.10 ppm) 1.1-2.5 (28H), 3.0-4.25 (88H), 4.5-5.2 (14H),7.3-8.02 (15H). ESI-MS m/z calcd for C₁₃₁H₁₉₁N₄O₇₀ 2940, found 2940[M]⁺.

<Test 6: Synthesis and Purification of Compound Represented by ChemicalFormula 25>

A mixture of 0.02 g of a compound represented by the chemical formula22, 0.1 g of a compound represented by the chemical formula 14, 0.032 gof WSC, 0.5 mL of pyridine and 0.05 mL of 0.1 M phosphoric acid bufferwas stirred at room temperature for 24 hours in a dark place. Afterthat, 10 mL of acetone was added thereto, and a precipitate was filteredunder a reduced pressure. The precipitate was dissolved in an aqueoussolution of 0.1% trifluoroacetic acid, and the solution was subjected toan ODS column chromatography. A compound represented by the chemicalformula 25 was eluted from the eluate using a mixed liquid of water andmethanol. The eluate was concentrated under a reduced pressure to obtain0.021 g of the green compound represented by the chemical formula 25 ina solid state.

Mechanical analysis data of the desired product represented by thechemical formula 25 are shown below. ¹H NMR (500 MHz, D₂O, 25° C.,Acetone: 2.10 ppm) 1.0-2.5 (32H), 3.0-4.5 (96H), 4.8-5.2 (14H), 6.09(2H, br), 6.37 (2H, br), 7.3-8.02 (15H, br). ESI-MS m/z calcd forC₁₃₇H₂₀₃N₄O₇₂ 3056, found 3056 [M]⁺.

<Test 7: Solubility of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention>

Solubility tests of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) in water or physiological saline were performed. About oneminute violent vibration-stirring was necessary for dissolving powderyICG from Molecular Probe Inc. in water or physiological saline. On theother hand, the vibration-stirring was not necessary for dissolving thecyclodextrin-bonded indocyanine compounds of the present invention, inparticular, the compounds represented by the chemical formula 16 and thechemical formula 20, and they were quickly dissolved.

<Test 8: Adsorption of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention to Human Skin>

Adsorption tests of ICG and the cyclodextrin-bonded indocyaninecompounds (the chemical formulae 16, 20, 21, and 23 to 25) of thepresent invention to human skin were performed. A 1 mM aqueous solution(0.03 mL) including ICG or each of the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) was put on an arm, and it was washed with water after 5minutes, rubbed it and further washed with water. The results were thatICG was not completely washed out with water; whereas, thecyclodextrin-bonded indocyanine compounds of the present invention, inparticular the compounds represented by the chemical formula 16 and thechemical formula 20 could be easily washed out. It was shown that theadsorption of the cyclodextrin-bonded indocyanine compound of thepresent invention to the human skin was much lower than that of ICG(FIG. 1).

<Test 9: Adsorption of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention to Cellulose Fiber>

Adsorption tests of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) to a cellulose fiber were performed. Using a cotton swab(Sanyo Co., Ltd.) as a cellulose fiber model, a 1 mM aqueous solution(0.05 mL) including ICG or each of the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) was applied to the model, and it was washed with runningwater (tap water, 1 L/minute) for 5 seconds after 3 minutes. The resultswere that ICG was not completely washed out with water; whereas, thecyclodextrin-bonded indocyanine compounds of the present invention, inparticular the compounds represented by the chemical formula 16 and thechemical formula 20 could be easily washed out. It was shown that theadsorption of the cyclodextrin-bonded indocyanine compound of thepresent invention to the cellulose fiber was much lower than that of ICG(FIG. 2).

<Test 10: Adsorption of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention to Meat Model of Living Body>

Adsorption tests of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) to a meat model of a living body were performed. Using acommercially available pork loin meat as the meat model of the livingbody, a depression part with a diameter of 5 mm was made on the porkloin meat, and a 1 mM aqueous solution (0.05 mL) including ICG or eachof the cyclodextrin-bonded indocyanine compounds of the presentinvention (the chemical formulae 16, 20, 21, and 23 to 25) was appliedto the depression part, and it was washed with running water (tap water,1 L/minute) for 10 seconds after 3 minutes. The results were that ICGwas not completely washed out with water; whereas, thecyclodextrin-bonded indocyanine compounds of the present invention, inparticular the compounds represented by the chemical formula 16 and thechemical formula 20, could be easily washed out. It was shown that theadsorption of the cyclodextrin-bonded indocyanine compound of thepresent invention to the meat model of the living body was much lowerthan that of ICG (FIG. 3).

<Test 11: Adsorption of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention to Protein Model of Living Body>

Adsorption tests of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) to a protein model of a living body. Using a commerciallyavailable chicken breast meat as the protein model of the living body, adepression part with a diameter of 5 mm was made on the chicken breastmeat, and a 1 mM aqueous solution (0.05 mL) including ICG or each of thecyclodextrin-bonded indocyanine compounds of the present invention (thechemical formulae 16, 20, 21, and 23 to 25) was applied to thedepression part, and it was washed with running water (tap water, 1L/minute) for 10 seconds after 3 minutes. The results were that ICG wasnot completely washed out with water; whereas, the cyclodextrin-bondedindocyanine compounds of the present invention, in particular thecompounds represented by the chemical formula 16 and the chemicalformula 20, could be easily washed out. It was shown that the adsorptionof the cyclodextrin-bonded indocyanine compound of the present inventionto the protein model of the living body was much lower than that of ICG(FIG. 4).

<Test 12: Adsorption of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention to Hydrophobic Chemical Fiber>

Adsorption tests of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) to a hydrophobic chemical fiber were performed. Using apolypropylene mask (Tamagawa-Eizai Co., Ltd.) as a model of hydrophobicchemical fiber, a 1 mM aqueous solution (0.05 mL) including ICG or eachof the cyclodextrin-bonded indocyanine compounds of the presentinvention (the chemical formulae 16, 20, 21, and 23 to 25) was appliedto the model, and it was washed with running water (tap water, 1L/minute) for one second after it was allowed to stand for 20 minutes(it was allowed to stand for 20 minutes for removing moisture, becauseif the sample has moisture, the compound is not adhered to the mask).The results were that ICG was not completely washed out with water;whereas, the cyclodextrin-bonded indocyanine compounds of the presentinvention, in particular the compounds represented by the chemicalformula 16 and the chemical formula 20, could be easily washed out. Itwas shown that the adsorption of the cyclodextrin-bonded indocyaninecompound of the present invention to the hydrophobic chemical fiber wasmuch lower than that of ICG (FIG. 5).

<Test 13: Molecule Association of Cyclodextrin-Bonded IndocyanineCompound of Present Invention in Aqueous Solution>

The molecule association of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) in an aqueous solution was studied. Each of 0.01 mM, 0.025mM, 0.05 mM, and 0.1 mM aqueous solutions including ICG or thecyclodextrin-bonded indocyanine compound of the present invention (eachchemical formula 16, 20, 21, and 23 to 25) was prepared, it was put in aquartz cell having a length of an optical path of 1 mm, and an opticalabsorption spectrum at 600 nm to 1000 nm was measured at 25° C. Theresults showed that molecule association, called as “H-aggregation,”occurred for ICG in this concentration range (FIG. 6, the left graph);whereas the molecule association, called as “H-aggregation,” did notoccur for the cyclodextrin-bonded indocyanine compounds of the presentinvention, in particular, the compounds represented by the chemicalformula 16 and the chemical formula 20, in this concentration range(FIG. 6, the right graph).

<Test 14: Fluorescence of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention in Aqueous Solution>

The fluorescence of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) in an aqueous solution was studied. A 0.1 μM aqueoussolution including ICG or each of the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) was put in a 1 cm square quartz cell at 25° C., which wasexcited with 720 nm excitation light (bandpass: 10 nm), and afluorescence spectrum (bandpass: 10 nm) was measured. A fluorescenceefficiency was calculated based on a fluorescence efficiency of ICG,0.13 (in DMSO, at 25° C.). The results were that ICG had a fluorescencequantum efficiency of 0.021; whereas the cyclodextrin-bonded indocyaninecompounds of the present invention, in particular, the compoundsrepresented by the chemical formula 16 and the chemical formula 20 hadfluorescence quantum efficiencies of 0.054 and 0.042, respectively. Thefluorescence quantum efficiencies thereof were respectively 2.6-fold and2-fold of that of ICG.

<Test 15: Fluorescence of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention in Blood>

The fluorescence of ICG and the cyclodextrin-bonded indocyaninecompounds of the present invention (the chemical formulae 16, 20, 21,and 23 to 25) in blood was studied. ICG and the cyclodextrin-bondedindocyanine compounds of the present invention (the chemical formulae16, 20, 21, and 23 to 25) were respectively dissolved in blood (human)in a concentration of 100 μM. The resulting blood was put in atriangular quartz cell at 25° C., which was excited with 760 nmexcitation light (bandpass: 10 nm), and a surface fluorescence spectrum(bandpass: 10 nm) was measured. ICG had a fluorescence intensity of 58(arbitrary unit) at the maximum fluorescence wavelength. Thecyclodextrin-bonded indocyanine compounds of the present invention, inparticular, the compounds represented by the chemical formula 16 and thechemical formula 20 had respectively fluorescence intensities of 270(arbitrary unit) and 190 (arbitrary unit), which were 4.7-fold and3.3-fold of that of ICG. It could be considered that this is causedbecause the emission of light from the cyclodextrin-bonded indocyaninecompound of the present invention was not hindered in the living body.

<Test 16: Behavior of Cyclodextrin-Bonded Indocyanine Compound ofPresent Invention in Living Body>

The behavior of the isomerization equilibrium compound (TK1) representedby the chemical formula 19 or 20 or the isomerization equilibriumcompound (TK2) represented by the chemical formula 15 or 16, which weretypical examples, was evaluated in human blood and a rat body, whichwere living bodies of interest.

Fluorescence Behavior in Human Blood

The concentration dependence in the surface fluorescence intensity ofICG, TK1, or TK2 was evaluated in human venous blood in a triangle cell.Specifically, 1.0 mL of human venous blood was put in a triangle quartzcell, and ICG, TK1 or TK2 was added thereto in a given concentration.Excitation light of 760 nm (bandpass: 10 nm) was irradiated thereto at25° C., and a surface fluorescence (bandpass: 10 nm) was measured. Theresults are shown in FIG. 7.

As apparent from FIG. 7, it was found that both TK1 and TK2 emittedfluorescence in the human venous blood by the irradiation of theexcitation light.

Study of Possibility of Administration into Rat Body

An aqueous solution including ICG, TK1, or TK2 in a concentration of 1mM was prepared, and 0.1 mL of the solution was injected into a femoralvein exposed in a rat which had undergone laparotomy. After that, 760 nmof excitation light was irradiated into the abdomen opened, and presenceor absence of fluorescence was evaluated.

As a result, the fluorescence was observed from internal organs in theabdomen opened. It was observed that the TK1 and TK2 were highlyaccumulated in kidney (as already known, ICG is highly accumulated inliver). Severe influences on the rat by the administration were notobserved, and it was confirmed that they could be administeredrelatively safely to the living body.

Observation of Distribution in Rat Body

After Wistar male rat (9-week old, a body weight of 350 g) wasether-anesthetized, 0.1 mL of Nembutal was intraperitoneallyadministered using a 26 G needle to perform the anesthesia. A base ofthe rat's tail was wrapped tightly with a rubber band to prevent theflow of blood, and the caudate vein was secured with a 24 G Therflowneedle and a peripheral line were secured by inserting a three-waystopcock and an extension tube. The rat was fixed on a flat stand in asupine position (FIG. 8). A PDE camera unit manufactured by HamamatsuPhotonics K.K. was set at exactly 16 cm from the body of the rat. Whenthe whole body was extensively photographed, the distance was 20 cm. Theobservation range was an about range shown by black dots in FIG. 8.After that, ICG, TK1 or TK2 was administered in a concentration of 1 mMfrom the peripheral line secured. The administration amount was 0.1 mL.After 20 minutes from the administration, the fluorescence state of theabdominal cavity which was opened is shown in FIG. 9, and thefluorescence state (images at the maximum luminance) of the dorsalregion of foot was shown in FIG. 12.

FIG. 9 shows that ICG was mainly accumulated in the liver (FIG. 9(a)),and TK1 and TK2 were mainly accumulated in the kidney (FIG. 9(b) andFIG. 9(C)). The near-infrared imaging of the internal organs immediatelyafter the administration showed that the kidney, ureter and bladder wereclearly imaged (FIG. 10). In addition, it also shown that the uretercould be imaged by the near-infrared endoscope (FIG. 11).

As apparent from the image view 12, the state in which the fluorescencewas emitted in accordance with the blood flow was clear in the dorsalregion of foot. It was also found that when ICG, TK1 or TK2 wasadministered as above and fluorescence was observed in an ischemia modelof a lower limbs which was caused by ligation of a right femoral arteryof a rat, the fluorescence was not observed in the ischemia area, andtherefore it was found that a degree of blood flow could be evaluatedbased on the presence or absence of the fluorescence, though it was notshown in a figure.

Here, the changes with time in the fluorescence intensity of the dorsalregion of foot were shown in FIG. 13 and FIG. 14. In addition, a timereaching the maximum fluorescence intensity value (Imax), Imax value anda time at which the value reached a half of the Imax value (t½) areshown in Table 1.

TABLE 1 Compound Time for I_(max) I_(max)*. t_(1/2) ICG 0.5 min 124 2.3min TK-1  10 min 160  40 min TK-2  15 min 106  55 min

As apparent from Table 1, FIG. 13 and FIG. 14, it was found that thetimes necessary for reaching the maximum fluorescence intensity valueand the times necessary for reducing it by half of TK1 and TK2 werelonger than those of ICG, in other words, they could emit thefluorescence for a long time in the body. It was found that, forexample, the fluorescence intensity of ICG was reduced to an aboutinitial value thereof in less than 10 minutes after the administration;whereas, TK1 and TK2 showed a high fluorescence intensity even after onehour from the administration.

Observation at Blood Vessel Level

ICG or TK1 was administered in a state in which a blood vessel of a ratwas directly exposed and observed by methods disclosed in Non PatentDocuments 7 and 8 (FIG. 15), and a state of a cremaster skin flap afterthe exposure is shown in FIG. 16. ICG or TK1 was administered in thestate in which the blood vessel was exposed, and the observation wasperformed. As a result, it was observed that ICG was unevenlydistributed in the blood vessel, compared to TK1. It was found thereforethat TK1 transferred from the blood vessel into the interstitial tissuefluid. In order to more precisely study it, photomicrographs of theblood vessel after the administration are shown in FIG. 17 (ICG) andFIG. 18 (TK1). As apparent from FIGS. 17 and 18, in comparison of themin the fluorescence intensity in the blood vessel and the fluorescenceintensity in the surrounding tissues thereof, the fluorescence washardly observed in the interstitial tissue for ICG; whereas thefluorescence was observed in the interstitial tissue for TK1, and theincrease of the fluorescence intensity with time was also observed inthe interstitial tissue, though it is not described in detail. It wasfound therefore that ICG hardly transferred from the blood vessel intothe interstitial tissue fluid; whereas TK1 transferred from the bloodvessel into the interstitial tissue fluid.

Evaluation of Relationship Between Degree of Transfer ofCyclodextrin-Bonded Indocyanine Compound of Present Invention intoInterstitial Tissue and Size of Edema Formed

(Evaluation Method)

Rats (male, Wister rat) which were 10-week old and about 350 g wereselected as a test animal (n=4). The rat was generally anesthetized withisoflurane, and 0.1 mL of 0.5% λ-carrageenin was injected to a leftposterior footpad using a 26 G needle, thus resulting quick generationof edema on the foot. After 15 minutes, a volume of the left foot of therat was measured using a rat footpad volume-measuring device (MK-101 CMPPLETHYSMOMETER manufactured by Muromachi Kikai Co., Ltd.).

After that, the isoflurane was off and the emergence of the rat wasconfirmed. After Von Frey test was performed, the rat was generallyanesthetized with isoflurane again. The caudate vein of the rat wassecured with a 24 G Therflow needle, and then it was fixed on ahorizontal stand in a supine position. A PDE camera manufactured byHamamatsu Photonics K. K. was set and fixed at a height of 16 cm from adorsalis pedis of the rat.

A moiety (ROI) to be measured by the PDE camera was set at a centralpart of the dorsalis pedis and light was turned off. After themeasurement was started, 0.1 mL of an aqueous solution including 1 mmolTK1 was infused from a caudate vein, and 1 mL of physiological salinewas flushed over 5 seconds. A continuous imaging was performed forinitial 5 minutes after the infusion to measure luminances in the ROI.After that, luminance was measured for one minute at 5 minute-intervalsand the measurement was continued until 120 minutes from the infusion.After the measurement was finished, the isoflurane was off to bring therat out of the anesthesia, and it was returned into a cage (acuteinflammation experiment).

After 7 days, a Von Frey test was performed and then the rat was quicklygenerally anesthetized with isoflurane, and the volume of the left footof the rat was measured by using the rat footpad volume-measuringdevice. The caudate vein of the rat was secured with a 24 G Therflowneedle, and then it was fixed on a horizontal stand in a supineposition. A PDE camera manufactured by Hamamatsu Photonics K. K. was setand fixed at a height of 16 cm from the dorsalis pedis of the rat. Afterthe ROI was set at the central part of the dorsalis pedis and light wasturned off, the measurement was started. An aqueous solution including 1mmol TK1, 0.1 mL, was infused from a caudate vein, and 1 mL ofphysiological saline was flushed over 5 seconds. A continuous imagingwas performed for initial 5 minutes after the infusion to measureluminances in the ROI. After that, luminance was measured for one minuteat 5 minute-intervals and the measurement was continued until 120minutes from the infusion. After the measurement was finished, theisoflurane was off to bring the rat out of the anesthesia, and it wasreturned into the cage. A group to which λ-carrageenin was not injectedwas subjected to the same test.

The same procedures as above were performed for ICG.

(Results)

The results are shown in FIGS. 19 to 26. The volume of the left foot ofthe rat in the group to which TK1 was administered are shown in FIG. 19(the higher the vertical axis, the larger the volume); the results ofthe Von Frey test are shown in FIG. 20 (the lower the vertical axis, thehigher the hyperalgesia); the change in the luminance immediately afterthe injection is shown in FIG. 21 (the higher the vertical axis, thehigher the luminance); and the change in the luminance after one weekfrom the carrageenin injection is shown in FIG. 23 (the higher thevertical axis, the higher the luminance). The volume of the left foot ofthe rat in the group to which ICG was administered is shown in FIG. 23(the higher the vertical axis, the larger the volume); the results ofthe Von Frey test are shown in FIG. 24 (the lower the vertical axis, thehigher the hyperalgesia); the change in the luminance immediately afterthe injection is shown in FIG. 25 (the higher the vertical axis, thehigher the luminance); and the change in the luminance after one weekfrom the carrageenin injection is shown in FIG. 26 (the higher thevertical axis, the higher the luminance). The values on the verticalaxes in all of the graphs are arbitrary units.

A large difference was observed between ICG and TK1 in the changepattern in luminance in the ROI. The luminance was increased in a shorttime after the administration of ICG, and then it was quickly turnedinto decrease and decreased to the base line in about 10 minutes. On theother hand, the TK1 showed the similar trajectory to that of ICG in theincrease of the luminance in the early state after the administration,but after that the luminance was stopped at a high level, and it took 6hours to return to the base line. From these results, it was consideredthat ICG stayed in the blood vessel without leakage into theinterstitial tissue, and the passage in the blood vessel distributed inROI caused the quick increase of the luminance and the subsequent quickdecrease thereof. On the other hand, it could be considered that TK1reflected two phases of a part in which the quick passage in the bloodvessel at the first stage was reflected (hereinafter referred to as a“blood vessel phase”) and a phase in which TK1, which gradually leakedto the interstitial tissue after that, emitted the fluorescence(hereinafter referred to as an “interstitial tissue phase”). A timenecessary for transfer from the blood vessel phase to the interstitialtissue phase corresponds to a predetermined time.

In the acute inflammation experiment, the remarkable increase of thefoot volume of the rat was observed after the λ-carrageenin injection inboth TK1 and ICG groups, and the volume was normalized to that at atendon side after one week (FIGS. 19 and 23). In the Von Frey test, theremarkable hyperalgesia was observed after the λ-carrageenin injectionin both TK1 and ICG groups, and the hyperalgesia was normalized to thatat the tendon side after one week (FIGS. 20 and 24).

It was observed that in the luminance of the dorsalis pedis, theluminance change in the TK1 group was not different from that of thecontrol group in the blood vessel phase, but the faster luminanceincrease in the λ-carrageenin administration group was observed in theinterstitial tissue phase, and it was observed TK1 group maintained highvalues during the evaluation time (FIG. 21). After one week from theλ-carrageenin administration, at which the inflammation caused by theadministration disappeared, difference in the luminance change was notobserved between the carrageenin administration side and the tendon sidein the interstitial tissue phase (FIG. 22). As clearly shown in thegraph of FIG. 21, the change in the luminance showed a smooth curve inthe interstitial tissue phase, and it was found that the height of thepeak depended on a rate of change of rising in the interstitial tissuephase, in other words, it shows that a change of values in theinterstitial tissue phase can be predicted in a high precision by a timeseries analysis, and shows important grounds for proving our workinghypothesis.

In ICG group, the difference in the luminance was not observed betweenthe immediately after the λ-carrageenin injection and after one weekfrom the administration (FIG. 25 and FIG. 26). From this result, it wasfound that ICG, which is highly hydrophobic and has hitherto beenclinically used, did not cause the leakage of the fluorescence substanceout of the blood vessel, even if there was a severe acute inflammationcaused by, for example, λ-carrageenin.

From the results described above, it became apparent that when thefluorescence intensity of the moiety in which tumescence progression ispredicted is measured with time during a term corresponding to theinterstitial tissue phase after TK1 was administered to a living body,the change in the subsequent fluorescence intensity can be predicted,and the change in the fluorescence intensity is relevant to thetumescence progression in that moiety. It can be found accordingly thatTK1 can be used in the prediction of tumescence which cannot bepredicted by using ICG.

INDUSTRIAL APPLICABILITY

According to the cyclodextrin-bonded indocyanine compound represented bythe chemical formula 1 or chemical formula 2 of the present invention, acompound which is a green pigment and emits near-infrared fluorescence,which characterized by having a higher solubility in water orphysiological saline, easier removal from biological tissues, lowermolecule association in an aqueous solution, and higher near-infraredfluorescence intensity in an aqueous solution compared to ICG, andincluding no iodine can be provided. Also according to the synthesismethod of the cyclodextrin-bonded indocyanine compound of the presentinvention, a useful synthesis of the cyclodextrin-bonded indocyaninecompound can be provided. Further according to the purification methodof the cyclodextrin-bonded indocyanine compound of the presentinvention, a useful purification of the cyclodextrin-bonded indocyaninecompound can be provided. Furthermore, the cyclodextrin-bondedindocyanine compound of the present invention can provide a diagnosticcomposition including no iodine which causes iodine hypersensitivity,because the compound shows a sufficient solubility even if the iodine isnot included. The diagnostic composition shows a biohehavior differentfrom that of a conventional diagnostic composition including ICG alone,and thus various useful diagnosis methods and diagnosis devices can beprovided utilizing the properties.

1. A cyclodextrin-bonded indocyanine compound represented by thefollowing chemical formula 1:

wherein R₁, R₂, R₃, R₄, R₅, R₆, R⁷, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄,R₁₅, R₁₆, R¹⁷, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃ are a hydrogen atom, analkyl group, an aryl group, a halogen atom, an alkoxyl group, an aminogroup, a carboxyl group, a formyl group, a sulfonyl group, a sulfonicacid group, a phosphate group, an alkyloxycarbonyl group, anaryloxycarbonyl group, an alkylcarbonyl group, an arylcarbonyl group, ora heterocyclic ring; when a hydrogen ion on the substituents (thecarboxylic acid, the sulfonic acid and the phosphoric acid) dissociates,a metal ion such as a sodium ion, a potassium ion, a magnesium ion or acalcium ion may be substituted for the hydrogen ion; the amino group isalso selected from primary, secondary, tertiary and quaternary groups; acyclic structure of CH₂, CH₂CH₂, CH₂CH₂CH₂ or CH₂CH₂CH₂CH₂ is alsoselected as the groups R₈ and R₉; and a functional group of an alkylgroup, an aryl group, a halogen atom, an alkoxy group, an amino group, acarboxyl group, a formyl group, a sulfonyl group, a sulfonic acid group,a phosphate group, an alkyloxycarbonyl group, an aryloxycarbonyl group,an alkylcarbonyl group, an arylcarbonyl group or a heterocyclic ring isalso substituted for the hydrogen atom on the alkyl groups. 2-25.(canceled)